Nucleic acid detection in a PCR by means of a target-sequence-unspecific modular reporter complex and electrochemical detection

Abstract

The invention relates to a method for detecting at least one target nucleic acid sequence by means of a method for detecting at least one target nucleic acid sequence, said method comprising the steps of: i. providing at least one target-sequence-unspecific modular reporter complex comprising at least one marker, and at least two oligonucleotides, namely i. a base strand comprising 1. at least one mediator binding site, and 2. at least one signal initiation oligo binding site, ii. at least one signal initiation oligo, the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo optionally being hybridised to one another but not covalently bonded and together forming a signal complex; j. providing at least one mediator probe, the mediator probe comprising an oligonucleotide having at least one probe sequence and at least one mediator sequence, the at least one probe sequence having an affinity for at least one target nucleic acid sequence, and the at least one mediator sequence having an affinity for at least one mediator binding site on the base strand of the at least one target-sequence-unspecific modular reporter complex; k. PCR amplification of at least one nucleic acid sequence; l. binding a probe sequence of at least one mediator probe to at least one target nucleic acid sequence; m. using a PCR polymerase to cleave the probe sequence of the at least one mediator probe that is bound to the at least one target nucleic acid sequence, the mediator sequence being released; n. binding at least one released mediator sequence to a mediator binding site of the at least one target-sequence-unspecific modular reporter complex; o. using a PCR polymerase to extend the sequence of at least one mediator sequence that is bound to a mediator binding site, the binding being broken or prevented by hybridising the at least one signal initiation oligo binding site and the at least one signal initiation oligo, thereby initiating a signal change; p. detecting at least one signal change as evidence of the at least one target nucleic acid sequence.

Description

[0001] Nucleic acid detection in a PCR using a target-nonspecific modular reporter complex and electrochemical de¬

[0002] TECTION

[0003] Technical background

[0004] In quantitative detection reactions of PCR products such as real-time PCR (qPCR) or digital PCR (dPCR), target sequences are primarily detected either by dyes that intercalate into the DNA, but bind non-specifically to all existing DNA double strands, or by DNA probes that bind only to a specific DNA target sequence. These DNA probes generate an optical signal change directly (e.g. Taqman probes) or indirectly (mediator probes in combination with universal reporter molecules) through their cleavage. Optical detectors detect the light emissions generated outside the reaction vessel during the reaction. These detection systems usually rely on light-absorbing and emitting fluorescent molecules. After excitation by light energy of a specific wavelength, these molecules release energy in the form of higher wavelengths, which can be detected by detectors.A distinction is made between molecules whose light energy is to be detected in a specific wavelength range (fluorescence donor or fluorophore) and molecules that lead to a decrease in the fluorescence intensity of a fluorophore upon spatial proximity (fluorescence acceptor or quencher). If the spatial proximity between fluorophore and quencher changes, the fluorescence signal changes accordingly, with a shorter distance always resulting in greater quenching (FRET quenching or contact quenching). The use of target sequence-specific DNA probes in combination with different fluorescent labels further enables the sensitive detection of multiple DNA sequences in a single reaction (multiplex detection).This typically involves the use of multiple DNA probes or detection molecules containing different fluorophore-quencher combinations, each of which emits a fluorescence signal in a specific wavelength range in the presence of a specific DNA sequence. Further developments of this multiplexing detection method also enable the detection of multiple target sequences within a single wavelength range by quantitatively analyzing the fluorescence values ​​or combining them with other parameters (e.g., the readout temperature).

[0005] Two well-known single-part target sequence-specific DNA probes for optical detection are Taqman probes and molecular beacons (Tan et al. 2004; Li et al. 2008; Holland et al. 1991; Rodriguez et al. 2005). These bind to the target sequence specifically and are cleaved during PCR, generating a signal. The disadvantage of these probes is their dependence on the target gene sequence, which means that only specific regions of a DNA target sequence that must meet certain requirements can be used. For example, attention must be paid to probe length, melting temperature, binding enthalpy, GC content, guanine quenching, and complementary sequence fragments. In addition, their synthesis can be very demanding. There are isolated examples of (specific) electrochemical detection of target sequences in the liquid phase. More frequently, the detection of target sequences by hybridization reactions on functionalized electrodes has been described.

[0006] Examples of liquid-phase detection utilize the molecular beacon method. Molecular beacons consist of an oligonucleotide (oligo) with five to seven complementary bases at each end and a terminal fluorophore or quencher (Tyagi and Kramer 1996). Instead of a fluorophore in a molecular beacon, an electroactive label is used, and instead of the quencher, a molecule complexes the electroactive molecule, thus preventing effective electron transfer. The complexing molecule can be omitted if the secondary structure of the molecular beacon forms a reversible binding region (in the form of an aptamer) for the electroactive molecule. In this case, hybridization of the target sequence with the molecular beacon causes the release of the electroactive molecule.

[0007] The specific electrochemical detection of target DNA is often performed using a solid phase, using specifically functionalized electrodes to which the amplification product or a probe hybridizes. Before the hybridization reaction, single-stranded DNA molecules are typically generated from double-stranded amplification products, for example, by enzymatic digestion. To generate the signal, either intercalating electroactive molecules or another hybridization probe modified with an electroactive molecule is then added.

[0008] DNA detection reactions can be found in the literature for which electrode surfaces have been functionalized with universally usable DNA probes. However, the target DNA is usually not amplified, resulting in low sensitivity. Some methods require the target sequence to be present as a single strand, which either requires an initial single-strand generation step or limits application to single-stranded RNA. Reaction times are also often significantly longer than expected for a DNA amplification reaction. To the best of our knowledge, only one form of hybridization chain reaction is comparable to, for example, PCR, for which, according to publications, detection limits of 50 aM are achieved within 120 min (Liu et al. 2017). This also requires a single-stranded target sequence that unfolds a first DNA probe (folded in a hairpin structure).When unfolded, the first probe can unfold a second universal probe (also initially present as a hairpin structure). This second probe is modified with an electroactive molecule. In the unfolded state, this second probe can hybridize to another surface-bound probe, thus accumulating electroactive molecules at the electrode surface.

[0009] Patent WQ2013079307A1, "Bifunctional Oligonucleotide Probe for Universal Real-Time Multianalyte Detection," claims the mediator probe technology system. In this system, a mediator probe is activated by extending a primer (auxiliary molecule 1) at the target sequence using a polymerase (auxiliary molecule 2). Due to the exonucleation activity of the polymerase, the mediator probe is cleaved and can then bind to the UR (mediator hybridization sequence). After cleavage, the mediator probe acts as a primer. This is then extended by the polymerase, separating the fluorophore and quencher on the universal reporter, resulting in signal generation.An example of a two-part reporter system can be found in patent WO2018114674A1 for the loop-mediated isothermal amplification method with mediator-displacement probes (MD LAMP). A universal reporter with at least one oligonucleotide and at least one fluorophore and quencher is claimed for a LAMP reaction. Until now, it was assumed that this only works because, unlike PCR, LAMP continuously maintains a uniform temperature, which quickly establishes and maintains equilibrium, allowing individual molecules to bind permanently to one another and thus generate no signal in the initial state.

[0010] Regarding detection on a solid phase, only single-part universal reporters bound to electrode surfaces have been described so far. The signal change is currently achieved by hybridization of a displaced mediator.

[0011] In multiplexed variations, different wavelengths are generally used in optical detection reactions to distinguish individual target sequences in specific light ranges, the so-called detection channels, within a reaction. This limits multiplexing, as only one target sequence can be detected per channel. Thus, the degree of multiplexing in most commercial devices is limited to five or six channels.

[0012] An alternative approach to multiplexing is represented by patents with detection systems that also rely on a separation of signal generation and detection, such as Seegene's patent US20200087718A1 for signal molecule-based detection using melting curve analysis. This patent claims the possibility of increasing the degree of multiplexing by using different lengths of the target sequence-unspecific signal molecule to generate different melting temperatures. A further divisional application (EP2708608), unlike the original patent, only uses signal readout at predefined temperatures, since the corresponding single-unit signal molecules of different sequence lengths also exhibit different fluorescence behavior at defined readout temperatures.

[0013] Another patent is the patent granted only in the USA by Biorad (US9921154B2). This also claims the detection of multiple target sequences in identical detection channels in a digital PCR, but according to current understanding, it can only be applied if there is a significant multiple occupancy of DNA target sequences in each reaction chamber of a digital PCR, thus leading to distinguishable signal clusters in the data space. The inventive method described here has the advantage over this patent that it can also be applied without multiple occupancy.

[0014] An example of universal electrochemical detection can be found in patent DE102011056606B3 from FRIZ Biochem GmbH, which describes a universal detection method for electrochemical detection. It uses a so-called signal oligo, which has a hairpin structure. The loop region contains a target DNA-specific sequence. The stem (or "star") of the hairpin contains a universal sequence that can hybridize to the complementary universal solid-phase probe and is modified with an electroactive molecule for detection. In the folded state, hybridization is not effective. When the signal initiation oligo hybridizes with the target sequence, it unfolds, and the sequences of the stem form flaps. During a DNA amplification reaction, the exonuclease domain of the polymerase cleaves the flap sequence, allowing it to hybridize more effectively with the solid-phase probe.

[0015] A disadvantage of this method is that the signal oligo is modified with a relatively expensive electroactive molecule. Since the signal oligo also contains a target sequence-specific region, assay optimization can be expensive. The design of the signal oligo and solid-phase probe is not trivial. By optimizing the melting temperatures, it is necessary to prevent the solid-phase probe from interacting with the still-folded signal oligo. At the same time, it is necessary to prevent the complementary parts of the stem from rehybridizing with each other even after cleavage by the exonuclease activity. The patent therefore describes that the solid-phase probe should contain individual bases that are not complementary to the stem of the signal oligonucleotide in order to optimize the melting temperatures.

[0016] Object of the invention

[0017] PCR is the gold standard method for amplifying individual DNA sequences and thus making them detectable. Either intercalating dyes or DNA probes are used to detect and quantify PCR products (DNA or cDNA in the case of RNA). However, intercalating dyes bind non-specifically to all existing double-stranded DNA molecules, thus preventing direct sequence-specific detection in PCR. DNA probes are signal-generating DNA sequences that are complementary to the respective sequence segment of a PCR product and can thus specifically detect and quantify it. This method is used in particular in real-time PCR and digital PCR. Currently, DNA probes either have a biochemical modification themselves to generate a signal in the presence of a DNA target sequence during PCR (e.g.Taqman probes) or activate a second detection molecule, which generates a signal independent of the target sequence (e.g. mediator probes in combination with target sequence-unspecific universal reporters).

[0018] For detection, the method according to the invention preferably uses electrochemical methods to detect DNA sequences in a PCR. These methods differ significantly from the previously described optical methods, as a signal change is generated via a change in the electrical charge transfer at an electrode. This means that the signal change must be detected at the electrode solid phase. This brings with it many advantages. For example, signal detection is very sensitive and direct, which eliminates the need for expensive optical detection systems. This is advantageous because corresponding electrochemical detectors are significantly cheaper, more robust, and easy to miniaturize. To date, the non-direct transferability between optical and electrochemical detection has been a problem, as it is not sufficient to simply add a biosensor (e.g., a DNA sequence) to an electrode.Rather, all detection components must be adapted and other detection mechanisms must be used. This hurdle prevents the rapid transfer of detection systems between optical and electrochemical detection. This highlights the urgent need for a new type of detection process and signaling molecules that overcome these problems. Furthermore, this detection process should be based on a general basic process so that similar DNA sequence layouts can be used for optical or electrochemical detection, ensuring rapid transfer between different detection methods.

[0019] The core of such a process would be a new type of modular detection molecule complex that is target sequence-nonspecific, flexible in design, and easy to optimize, while possessing the same performance characteristics as the current state of the art for single-part detection molecules. To achieve this, it should possess similar sequence patterns to facilitate the transfer of detection processes between optical and electrochemical detection. To date, such modular compositions have failed, particularly because they lack covalent chemical bonds, which, according to current state-of-the-art assumptions, makes them unsuitable for PCR applications.

[0020] Once optimized, the reaction of the signal-causing or signal-initiating oligonucleotides, particularly the hybridization reaction on the solid phase surface, should also be easily and reproducibly transferable to other target sequences. The universal nature of the surface modification should allow the sequences involved in the surface reactions to remain unchanged, even if these surfaces are to be used to detect additional target sequences. The target sequence-specific reactions should therefore all take place in the liquid phase, so that for the development of a new nucleic acid detection method, only the reagents and oligonucleotides of the liquid phase need to be adapted, redeveloped, and optimized. The surfaces and their modifications (e.g., the immobilization of oligonucleotides on electrode surfaces), on the other hand, should be producible in large batches and thus cost-effectively.These measures are intended to save development effort (time and costs).

[0021] Summary of the invention

[0022] The object of the invention is achieved by the features of the independent claims. Advantageous embodiments of the invention are described in the dependent claims.

[0023] Therefore, in one aspect, the invention relates to a method for detecting at least one target nucleic acid sequence, comprising the steps of: a. Providing at least one target sequence-unspecific modular reporter complex comprising at least one label, and at least two oligonucleotides, namely i. a base strand comprising

[0024] 1. at least one mediator binding site

[0025] 2. at least one signal initiation oligo binding site ii. at least one signal initiation oligo, wherein optionally the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo are hybridized to each other, but not covalently linked, and together form a signal complex. b. providing at least one mediator probe, wherein the mediator probe comprises an oligonucleotide with at least one probe sequence and at least one mediator sequence, wherein the at least one probe sequence has an affinity for at least one target nucleic acid sequence, and the at least one mediator sequence has an affinity for at least one mediator binding site on the base strand of the at least one target sequence-non-specific modular reporter complex, c. PCR amplification of at least one nucleic acid sequence, d. binding a probe sequence of at least one mediator probe to at least one target nucleic acid sequence, e.Cleavage of the probe sequence of the at least one mediator probe bound to the at least one target nucleic acid sequence by a PCR polymerase, whereby the mediator sequence is released, f. Binding of at least one released mediator sequence to a mediator binding site of the at least one target sequence-non-specific modular reporter complex, g. Extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, whereby the bond between the hybridized at least one signal initiation oligo binding site and the at least one signal initiation oligo is broken, whereby a signal change is initiated, h. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence.

[0026] In a further preferred embodiment, the method for detecting at least one target nucleic acid sequence comprises the following steps: a. Providing at least one target sequence-unspecific modular reporter complex comprising at least one label, and at least two oligonucleotides, namely i. a base strand comprising

[0027] 1. at least one mediator binding site

[0028] 2. at least one signal initiation oligo binding site ii. at least one signal initiation oligo, wherein optionally the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo are hybridized with each other, but are not covalently linked and together form a signal complex, b. providing at least one mediator probe, wherein the mediator probe comprises an oligonucleotide with at least one probe sequence and at least one mediator sequence, wherein the at least one probe sequence has an affinity for at least one target nucleic acid sequence, and the at least one mediator sequence has an affinity for at least one mediator binding site on the base strand of the at least one target sequence-non-specific modular reporter complex, c. PCR amplification of at least one nucleic acid sequence, d. binding a probe sequence of at least one mediator probe to at least one target nucleic acid sequence, e.Cleavage of the probe sequence of the at least one mediator probe bound to the at least one target nucleic acid sequence by a PCR polymerase, whereby the mediator sequence is released, f. Binding of at least one released mediator sequence to a mediator binding site of the at least one target sequence-non-specific modular reporter complex, g. Extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, whereby the binding of the at least one signal initiation oligo binding site and the at least one signal initiation oligo is broken and / or prevented, preferably via hybridization, thereby initiating a signal change, h. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence.

[0029] In a further preferred embodiment, the method for detecting at least one target nucleic acid sequence comprises the following steps: a. Providing at least one target sequence-unspecific modular reporter complex comprising at least one label, and at least two oligonucleotides, namely i. a base strand comprising

[0030] 1. at least one mediator binding site

[0031] 2. at least one signal initiation oligo binding site ii. at least one signal initiation oligo, wherein optionally the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo are hybridized with each other, but are not covalently linked and together form a signal complex, b. providing at least one mediator probe, wherein the mediator probe comprises an oligonucleotide with at least one probe sequence and at least one mediator sequence, wherein the at least one probe sequence has an affinity for at least one target nucleic acid sequence, and the at least one mediator sequence has an affinity for at least one mediator binding site on the base strand of the at least one target sequence-non-specific modular reporter complex, c. PCR amplification of at least one nucleic acid sequence, d. binding a probe sequence of at least one mediator probe to at least one target nucleic acid sequence, e.Cleavage of the probe sequence of the at least one mediator probe bound to the at least one target nucleic acid sequence by a PCR polymerase, whereby the mediator sequence is released, f. Binding of at least one released mediator sequence to a mediator binding site of the at least one target sequence-non-specific modular reporter complex, g. Extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, whereby the binding or hybridization of the at least one signal initiation oligo binding site and the at least one signal initiation oligo is either i) broken and / or prevented or ii) initiated and / or produced, whereby a signal change is initiated, h. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence.

[0032] In embodiments of the method according to the invention (e.g. similar to that shown in Figure 6 ii)), the signal oligo and the base strand are not hybridized with each other from the outset and do not yet form a signal complex in step a. The extension of the mediator on the base strand can, in some such embodiments, e.g., take place in a separate chamber / cavity / well, so that the hybridization, for example in a (subsequent) endpoint detection, only takes place at a later point in time / in a later step, quasi retrospectively. In some of these embodiments, the signal change in step g. is therefore not a “signal change” but rather an altered signal compared to a control reaction with an intact mediator probe. In embodiments of the method according to the invention, the signal change in step g.an altered signal, preferably an altered signal compared to a control reaction with uncleaved mediator probe, that is initiated or present.

[0033] In embodiments, step g. comprises extending the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, wherein the binding (and / or hybridization) of the at least one signal initiation oligo binding site and the at least one signal initiation oligo is either i) disrupted and / or prevented, or ii) initiated or enabled / produced, thereby producing an altered signal compared to a control reaction with uncleaved mediator probe.

[0034] In embodiments, step g comprises extending the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, wherein the binding (hybridization) of the at least one signal initiation oligo binding site and the at least one signal initiation oligo is either i) disrupted and / or prevented, or ii) initiated or enabled / produced, thereby initiating a signal change. In embodiments, a signal change is an altered signal, preferably an altered signal compared to a control reaction with uncleaved mediator probe.

[0035] Exemplary embodiments are shown in Figures 6i-6v. In some embodiments, a signal change is generated by the hybridization of a signal initiation oligo with an oligonucleotide, wherein preferably one of the two molecules comprises at least one label; in other embodiments, a signal change is generated by the prevention or cleavage / breaking of hybridization. In certain embodiments, a signal change is achieved by changing the distance of at least one electroactive molecule from a surface as a result of the severing of the bond between the signal initiation oligo and the base strand. In this case, extending the mediator either causes the distance from the surface to increase (Figure 6iv) or decrease (Figure 6v), which in both cases leads to a signal change.

[0036] In embodiments, step g. comprises extending the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, wherein the binding of the, optionally hybridized, at least one signal initiation oligo binding site and the at least one signal initiation oligo is disrupted and / or prevented, thereby initiating a signal change and / or a signal which is altered (e.g., compared to a control reaction).

[0037] In embodiments, in step a), the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo are hybridized to each other, but not covalently linked, and together form a signal complex.

[0038] In embodiments of the method according to the invention, a signal change is initiated in step g. by the separated at least one signal initiation oligo comprising at least one label and binding to at least one auxiliary oligo (preferably comprising a signal oligonucleotide binding site) which is immobilized on a solid phase. Non-imitating embodiments of the embodiments are shown in Figure 6 iii). In embodiments of the method according to the invention, a signal change is initiated in step g. by one of the two oligonucleotides or both oligonucleotides of the at least one target sequence-nonspecific modular reporter complex, namely i. the base strand, and / or ii. the at least one signal initiation oligo, being immobilized on a solid phase via a linker.

[0039] The modular reporter system for target sequence non-specific detection is a new invention that distinguishes itself from previously known detection systems through the flexible use of various oligonucleotides with different labels. The modular design, in particular, offers various advantages, such as the flexible adaptability to specific instrumentation requirements, the uniformity of the signal generation reaction between different detection methods, and the inexpensive, simple production, as well as entirely new multiplex detection methods. The system and its functionality have not yet been described in a patent or in the literature on nucleic acid detection in PCR.

[0040] The core of the invention is the modular, target-sequence-independent modular reporter complex (e.g., in Figure 1) composed of non-covalently linked oligonucleotides. This system possesses all the advantages of various target-sequence-specific detection systems and non-target-sequence-specific detection molecules, combining them with increased flexibility in the design of such detection molecules, leading to entirely new detection methods. Surprisingly, the system can be used without difficulty in a PCR with cyclic temperature changes, and the associated melting of the DNA strands in each cycle. Investigations by the inventors have shown that the modular system according to the invention even surpasses the functionality of prior art systems. This allows for significantly lower production costs while maintaining at least equally good performance parameters.A further advantage of the system according to the invention is the possibility of flexible adaptation of the universal probe according to the invention.

[0041] The modular system of the target sequence-independent modular reporter complex according to the invention consists of a basic scaffold consisting of a base strand. This has at least one binding site for a mediator (receptor complex) and at least one binding site for a signal initiation oligonucleotide (signal complex). The base strand and at least one signal initiation oligonucleotide together form a complex (e.g., Figure 1). At least one of these strands has a label that can detect a structural change in this complex. The modular structure results from several possible signal initiation oligonucleotides (or "signal initiation oligos"), which can be combined with a base strand in different ways in different embodiments (see, for example, Figures 5-7). Through various possibilities for attaching signal initiation oligonucleotides and base strands to a solid phase (e.g.,With the use of an electrode, the advantages of state-of-the-art target gene-dependent systems can be leveraged. The modular design allows for a much higher number of labels to be applied per complex than is possible with current single-component detection molecules. This novelty of the modular detection complex opens up new possibilities for detection reactions based on a basic reaction, which is described as an example below.

[0042] During a PCR detection reaction, a Mediator probe binds to the amplified DNA target sequence (Figure 1). A Mediator probe is an oligonucleotide and has a sequence-specific probe segment that binds to the target sequence and, in some preferred embodiments, is protected at the 3' end, and a target-sequence-nonspecific segment, called the Mediator, which does not bind to the target sequence except for one nucleotide that is common to both Mediator and probe. During primer extension by a polymerase with exonuclease activity, the Mediator is cleaved from the probe, leaving the common base on the Mediator (Figure 1). The Mediator is now no longer blocked by the probe segment and can bind to the target-sequence-nonspecific reporter complex and be extended there. The Mediator binds to the receptor complex of the base strand of the target-sequence-independent modular reporter complex.Subsequently, the mediator is extended along the base strand by the polymerase, whereby the signaling complex is broken down in such a way that individual components and / or molecules of this complex are cleaved off, thereby initiating a signal change compared to the original state (Figures 5-7).

[0043] The present invention achieves the advantageous effect that a once optimized reaction of the signal-causing or -initiating oligonucleotides, in particular the hybridization reaction on the solid phase surface, can be easily and reproducibly transferred to other target sequences. A further advantage of the invention is that the universal nature of the surface modification allows the sequences involved in the surface reactions to remain unchanged, even if these surfaces are to be used for the detection of other target sequences. In embodiments, a target sequence-specific reaction preferably takes place in the liquid phase, so that for the development of a new nucleic acid detection method, only the reagents and oligonucleotides of the liquid phase need to be adapted, newly developed, and optimized. Furthermore, it is advantageous that preferably the surfaces and their modification (e.g.The immobilization of oligonucleotides on electrode surfaces can be produced in large batches and thus cost-effectively. In summary, the invention enables development efforts (time and costs) to be saved.

[0044] In embodiments of the method according to the invention, either the base strand or the at least one signal initiation oligo comprises the at least one label.

[0045] To detect an electrochemical signal change during an amplification reaction, cleavage of the mediator probe and subsequent extension of the mediator on the target sequence-unspecific modular reporter can disrupt the bond of an electroactively labeled signal initiation oligo to the base strand. This oligo can then bind to a temporary oligo immobilized on a solid phase (electrode), thus inducing a signal change (Figure 6iii). As an alternative approach, the target sequence-independent modular reporter complex can be coupled to a solid phase via a linker on the base strand (Figure 6i). By extending the mediator, the bond between the signal initiation strand and the electroactive label, or the bond between the electroactive label itself and the base strand, is disrupted, resulting in a signal change.As a further alternative, the signal initiation oligo itself can be immobilized to a surface, and the entire target sequence-unspecific modular reporter complex can be coupled to the electrode via the base strand, which in this case possesses an electroactive modification (Figure 6ii). By extending the mediator, the binding of the base strand to the signal initiation oligo is broken or prevented, resulting in a signal change.

[0046] The probability of hybridization to a solid-phase probe can be influenced by the effective free enthalpy of the hybridization reaction and / or by different diffusion rates of molecules of different sizes (length and secondary structure of the DNA (complex)). In general, it is possible to extend the method to other detection principles, in which the signal is detected by enrichment / depletion of signal-generating labels on the solid phase. Among other things, fluorophores could be used for optical detection or magnetic particles for magnetoresistive detection.

[0047] In embodiments of the method according to the invention, the signal change in step g is based on the fact that the extension of the mediator sequence leads to an increased or reduced probability of hybridization of the labeled oligonucleotide to the immobilized oligonucleotide and thus the labeling on the functionalized surface is enriched or reduced.

[0048] Non-imitating embodiments of the embodiments with an increased hybridization probability are illustrated in Figure 6 iii). Non-imitating embodiments of the embodiments with a reduced hybridization probability are illustrated in Figures 6 i)-ii).

[0049] In embodiments, the detection methods are selected from the group comprising electrochemical, optical, magnetoresistive, piezoelectric (e.g., quartz crystal, surface acoustic waves), and / or surface plasmon resonance detection.

[0050] In embodiments of the method according to the invention, the at least one label is or comprises at least one electroactive label and the solid phase is an electrode.

[0051] In embodiments of the method according to the invention, a signal change is initiated in step g. by increasing or decreasing the distance between the at least one label and the solid phase in step g. Non-imitating examples of these embodiments are illustrated in Figures 6iv) and v).

[0052] Another option is the immobilization of the electroactive label to the solid phase (electrode), either via the base strand (Figure 6iv) or the signal initiation oligo (Figure 6v). In this case, a signal change is achieved by changing the distance from the electroactive molecule to the surface as a result of the severing of the bond between the signal initiation oligo and the base strand. In this case, extending the mediator causes the distance to the surface to either increase (Figure 6iv) or decrease (Figure 6v), which in both cases leads to a signal change. These embodiments are also not limited to electrochemical detection. Changing the distance of a fluorescent label can lead to a detectable signal change, for example, through surface quenching effects.

[0053] Another variant is detection comparable to the optical embodiment shown in Figure 7. After hydrolysis of the labeled signal initiation oligonucleotide, the label exhibits a detectably higher diffusion rate and can thus be detected even without enrichment by an oligonucleotide immobilized on the electrode. This effect was described, for example, in Pearce, DM, Shenton, DP, Holden, J., Gaydos, CA, 2011. IEEE Trans. Biomed. Eng. 58 (3), 755-758. However, in the embodiment described there, the addition of an electroactively labeled hydrolysis probe after PCR is necessary, which is then digested by an exonuclease that must also be added.

[0054] The advantage of the invention is, firstly, the possibility of analyzing suitable embodiments for signal generation more flexibly and quickly using the target sequence-nonspecific reporter complex, thereby generating stronger and / or more precisely distinguishable signals and providing a uniform mechanism for electrochemical detection than is possible with the state of the art. With regard to electrochemical solid-phase detection, the invention also allows for the generation of universally nationalized electrode arrays. This eliminates the need to adapt the electrode modification to new target sequences, and the electrode arrays can be produced in large batches and thus more cost-effectively.

[0055] Therefore, in embodiments, the invention encompasses various detection methods, such as electrochemical, optical (e.g. by fluorescence, dyes, etc.), magnetoresistive, piezoelectric (e.g. oscillating quartz, surface acoustic waves) or surface plasmon resonance.

[0056] In embodiments of the method according to the invention, an optically detectable marker such as colloidal gold, colloidal silver, colloidal carbon, latex particles is coupled to the marking.

[0057] In embodiments of the method according to the invention, the signal change in step g. is based on the fact that the change in the number of bound labels results in a change in the charge transfer.

[0058] In embodiments of the method according to the invention, the signal change in step g. is based on the fact that the number of bound labels results in a change in the electrode potential or the charge transfer.

[0059] In embodiments of the method according to the invention, the described change is determined as a changed current or charge or potential or charge transfer resistance by means of, for example, voltammetric, amperometric, coulometric, potentiometric or impedimetric measuring methods.

[0060] Thus, in embodiments, the invention encompasses electrochemical detection methods. The person skilled in the art is familiar with the basic possibilities and principles of electrochemical detection. In embodiments of the method according to the invention, the at least one label comprises at least one fluorophore. In embodiments, the at least one label of an oligonucleotide comprises at least one fluorophore, with the oligonucleotide bound to the solid phase. In such embodiments, quenchers can preferably be omitted.

[0061] In some embodiments of the method according to the invention, the signal change is based on the fact that the number of bound labels results in a change in the fluorescence intensity.

[0062] In embodiments of the method according to the invention, the signal change is based on the fact that changing the distance between the label and the solid phase causes a signal change. The solid phase is a (preferably metallic) surface that causes fluorescence quenching of proximal fluorophores.

[0063] In embodiments of the method according to the invention, the at least one label is a molecule for coupling to further labels. Molecules that can be used for coupling are known to the person skilled in the art. Non-mimicking examples are biotin-streptavidin, fluorescein-anti-fluorescein, etc.

[0064] In embodiments of the method according to the invention, the signal change is an optically perceptible change (for example by means of the eye or camera) in the color intensity at the location of the immobilized oligonucleotides.

[0065] Thus, the invention encompasses optical detection methods in some embodiments. The basic possibilities and principles of optical detection are known to those skilled in the art.

[0066] In embodiments of the method according to the invention, a nanoparticle (preferably a metal nanoparticle) is coupled to the marker and the solid phase is a piezoelectric material that is excited to oscillate by an alternating electric field.

[0067] In some of these embodiments of the method according to the invention, the signal change in step g. is a change in the frequency and / or phase and / or damping of the oscillation depending on the number of bound labels.

[0068] Thus, the invention encompasses piezoelectric detection methods in some embodiments. The basic possibilities and principles of piezoelectric detection are known to those skilled in the art.

[0069] In embodiments of the method according to the invention, a metallic nanoparticle is coupled to the marker and the solid phase is a metallic surface.

[0070] In embodiments of the method according to the invention, the signal change is based on a change in the refractive index at the surface due to the increase / decrease of bound labels, which can be determined by surface plasmon resonance spectroscopy.

[0071] Thus, in some embodiments, the invention encompasses surface plasmon resonance detection methods. The person skilled in the art is familiar with the basic possibilities and principles of surface plasmon resonance detection. In embodiments of the method according to the invention, magnetic nanoparticles are coupled to the marker, and the signal change is based on a change in the resistance of a magnetoresistive sensor.

[0072] Thus, the invention encompasses magnetoresistive detection methods in some embodiments. The basic possibilities and principles of magnetoresistive detection are known to those skilled in the art.

[0073] In embodiments of the method according to the invention, the immobilized oligonucleotides can be immobilized at several defined positions on the solid phase. The immobilized oligonucleotides can differ in their sequence. In embodiments, the solid phase is or comprises an electrode array and / or solid phase array.

[0074] In embodiments of the method according to the invention, the target sequence can be uniquely identified by the characteristics of the label and / or the position of the signal change. If detection takes place using a solid-phase array in embodiments, the position of the detected signal can also enable identification of the target sequence.

[0075] In embodiments of the method according to the invention, the separated at least one signal initiation oligo comprises at least one electroactive label, and wherein in step g. the at least one electroactive label is released, whereby it has a detectably higher diffusion rate and thus initiates a signal change by increasing the electron transfer rate.

[0076] In embodiments of the method according to the invention, the at least one label is specific for the at least one target nucleic acid sequence and wherein the signal change initiated under step g. is characteristic of the at least one target nucleic acid sequence.

[0077] In embodiments of the method according to the invention, the detection of the signal change comprises an analysis of the signal change as a function of the detection temperature and / or the electric field and / or the frequency of the excitation signal.

[0078] In some preferred embodiments, a mediator probe comprises an oligonucleotide and a sequence-specific probe segment that binds to the target sequence and is protected at the 3' end. This protection at the 3' end can be a blocking group (protecting group), e.g., a chemical blocking or protecting group, which in some embodiments comprises a chain of three carbon atoms. Protection of the mediator probe at the 3' end preferably prevents (non-specific) extension of the sequence strand by a polymerase during an amplification reaction. According to the invention, in embodiments, the mediator probe can comprise any protecting or blocking group suitable for preventing (non-specific) extension of the mediator probe sequence strand by a polymerase during an amplification reaction.In some embodiments, the mediator probe is protected against (non-specific) polymerase extension by means other than a blocking group (protecting group) at the 3' end.

[0079] In other embodiments, a Mediator probe does not comprise a blocking group (protecting group) at the 3' end and is not protected against (non-specific) polymerase extension. In embodiments, a Mediator probe protected at the 3' end may comprise a "C3 spacer." Such a C3 spacer may be a chemical blocking group, which in some embodiments comprises a chain of three carbon atoms. This "C3 spacer" thus preferably prevents (non-specific) polymerase extension of the Mediator probe sequence strand. Typical blocking groups (protecting groups) that are suitable depending on the embodiment are known to those skilled in the art. Based on the present disclosure of the invention, those skilled in the art also know how to select suitable blocking groups (protecting groups) as routine adaptations of the invention described herein.

[0080] The inventive process for detecting DNA sequences using PCR in combination with electrochemical readout accordingly comprises a novel system of individual DNA oligonucleotides. These form such a target-sequence-independent modular reporter complex without covalent bonds. This target-sequence-independent modular reporter complex surprisingly possesses all the advantages and performance characteristics of single-part target-sequence-dependent DNA probes or single-part target-sequence-independent detection molecules. The target-sequence-independent modular reporter complex consists of at least two DNA sequences that bind specifically to one another and carry modifications that initiate signal generation during PCR. This makes the target-sequence-independent reporter molecules flexible in design.A further inventive feature is that the reporter complexes according to the invention enable optical and electrochemical readout, whereby only the types of modifications differ, not the basic concept of the detection process. This resulting target-sequence-independent modular reporter complex possesses very high stability under PCR conditions in its initial state. The initial signal is accordingly comparable to that generated by a single-unit DNA probe or a single-unit target-sequence-independent reporter. As a result, the entire resulting PCR detection system according to the invention possesses comparable performance characteristics to current PCR detection methods based on single-unit detection molecules, although this complex of a target-sequence-independent modular reporter must be separated with each PCR cycle and reformed before signal readout.The system according to the invention thus increases the efficiency of signal generation and is also suitable for the simultaneous detection of multiple DNA target genes in a single PCR reaction (multiplex PCR). Furthermore, the complex is flexible in design, and the individual components are inexpensive to develop and produce. Furthermore, the system enables the production of universal microarrays, for example, for electrochemical detection. This allows the functionalization of the solid phase to be carried out in a highly standardized manner and in large batches. The hybridization reaction on the solid phase itself is also reproducible after initial optimization, since the same oligonucleotide combinations are always involved, so that only the partial reactions in the liquid phase need to be optimized for new target sequences.This reduces the problem that otherwise parts of the (amplified) target sequence usually hybridize to a solid-phase probe, the properties of which (length, secondary structure, etc.) can influence the hybridization efficiency and thus the signal. The process according to the invention also does not require an additional step for generating a single strand from the (amplified) target sequence, as is often the case in the prior art. Overall, this represents a significant improvement over the prior art. The various applications described herein are possible because the target sequence-independent modular reporter complex according to the invention is both flexible in design and inexpensive to develop and produce. Furthermore, the system according to the invention enables the production of universal microarrays.Overall, this represents a significant improvement over the state of the art.

[0081] In a further aspect, the invention relates to a kit for carrying out the method according to one of the preceding claims, comprising: at least one oligonucleotide primer at least one mediator probe at least one signal initiation oligo at least one base strand at least one buffer

[0082] PCR polymerase.

[0083] In the context of the kit according to the invention, the oligonucleotide primers, preferably at least one oligonucleotide primer pair, the at least one signal oligo, the at least one mediator and / or the at least one base strand can be configured or suitable for the specific detection of one or more different target sequences.

[0084] In embodiments, the kit according to the invention can be used for carrying out the method according to the invention.

[0085] In some embodiments, the kit can thus be used for the specific amplification and / or detection of target sequences within the scope of the method according to the invention. In preferred embodiments of the use of the kit according to the invention, the specific amplification and / or detection reaction is a PCR, qPCR, real-time PCR, droplet PCR, and / or digital PCR.

[0086] The embodiments described for one aspect of the invention may also be embodiments of any of the other aspects of the present invention. Accordingly, embodiments described for the method according to the invention may also be embodiments of the kit according to the invention. Furthermore, each embodiment described herein may also comprise features of any other embodiment of the invention. The various aspects of the invention are united by, benefit from, are based on, and / or are linked to the common and surprising discovery of the unexpected advantageous effects of the present method, namely the optimized PCR detection of target sequences by reporter complexes which are themselves target sequence non-specific.

[0087] Detailed description The term “target sequence-nonspecific reporter complex” describes a complex of target sequence-nonspecific DNA oligonucleotides for signal generation during PCR in the presence of the DNA target sequences.

[0088] In the context of the present invention, the term "base strand" describes a DNA oligonucleotide and part of the target sequence-nonspecific reporter complex. A base strand serves as the basis for the binding of signal initiation oligonucleotides and mediators, which thus form a signaling complex and receptor complex. In the context of the present invention, the term "base strand" describes a nucleic acid oligonucleotide (e.g., a DNA oligonucleotide) and part of the target sequence-nonspecific reporter complex. A base strand serves as the basis for the binding of signal initiation oligonucleotides and mediators, which thus form a signaling complex and receptor complex. Therefore, a base strand preferably comprises at least one mediator binding site and at least one signal oligo binding site. In embodiments, a base strand comprises one or more mediator binding sites and / or signal oligo binding sites.Thus, a base strand can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or even 50 Mediator binding sites. A base strand can also comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or even 50 signal oligo binding sites. In preferred embodiments, a base strand comprises between 1 and 10 Mediator binding sites and between 1 and 10 signal oligo binding sites. A mediator binding site can correspond to one or more signal oligo binding sites, in other words, an (activated) mediator bound to a mediator binding site can, when extended by a (PCR) polymerase, lead to the activation or degradation, digestion, cleavage or release of one or more signal oligos from one or more, preferably upstream (towards the 5' end), signal oligo binding sites.A basic strand may comprise one or more signaling complexes and one or more receptor complexes, wherein a signaling oligo complex may comprise at least one signaling oligo binding site and (in the non-activated state) at least one signaling oligo, and a receptor complex may comprise at least one mediator binding site.

[0089] In the context of the present invention, a signal initiation oligonucleotide is a DNA oligonucleotide and part of the signaling complex, which initiates a signal change in the presence of a target sequence by itself and / or parts thereof being cleaved or broken down from the signaling complex.

[0090] In the context of the present invention, a "signal initiation oligonucleotide," "signal initiation oligo," or "signal oligo" for short, is a nucleic acid oligonucleotide (preferably a DNA oligonucleotide) and part of the signaling complex that initiates a signal change in the presence of a target sequence by itself and / or parts thereof being cleaved or broken apart from the signaling complex. In the context of the present invention, the terms signal initiation oligonucleotide, signal initiation oligo, and signal oligo are considered equivalent and interchangeable.

[0091] In the context of the present invention, a label may preferably be one or more electroactive labels or one or more fluorophores and / or a molecule for coupling further labels. Accordingly, in the context of the invention, a base strand and / or a signal initiation molecule or signal initiation oligo may, in embodiments, carry or comprise one or more electroactive labels and / or fluorophores and / or molecules for coupling further labels. Herein, a "mediator" refers to an oligonucleotide and part of the receptor complex that can be extended along the "base strand" by the polymerase.A “mediator probe” describes a DNA oligonucleotide that establishes the connection between the target sequence and the target sequence-independent (modular) receptor by binding to a DNA target sequence during PCR in the presence of the target sequence, being cleaved by the exonuclease activity of the polymerase, and releasing a mediator sequence that forms a receptor complex with the base strand.

[0092] The term "nucleic acid" refers to nucleic acid molecules, including, without limitation, DNA, ssDNA, dsDNA, RNA, mRNA, tRNA, lncRNA, ncRNA, microRNA, siRNA, rRNA, sgRNA, piRNA, rmRNA, snRNA, snoRNA, scaRNA, gRNA, or viral RNA. Nucleic acid sequences herein refer to a consecutive arrangement of nucleotides, where nucleotides are represented by their nucleobases in guanine (G), adenine (A), cytosine (C), and thymine (T) in DNA and uracil (U) in RNA. A nucleic acid sequence may also refer herein to the sequence of consecutive letters or nucleobases (consisting of G, A, C, and T or U) that represent the actual sequence of consecutive nucleic acids in a DNA or RNA strand. This nucleic acid sequence can be identified and characterized biochemically and bioinformatically using DNA or RNA sequencing or by complementary nucleic acid probes (e.g.In embodiments herein, mediator probes) are specifically detected, e.g., in the context of a PCR, real-time PCR, or digital PCR detection reaction. The sequence analysis can also comprise the comparison of the resulting nucleic acid sequence or a detection signal specific thereto with one or more reference nucleic acid sequences and / or with the detection signals of housekeeping genes. The term nucleotide can be abbreviated to "nt." The term base pair (two nucleobases bound to each other via hydrogen bonds) can be abbreviated to "bp."

[0093] In the context of the invention, a "target sequence" describes any nucleic acid sequence of interest that is to be detected by the method according to the invention. A target sequence can preferably be a DNA or RNA sequence. A target sequence can represent part or all of a target DNA nucleic acid sequence. A mediator probe preferably comprises a sequence that is fully or partially complementary to the nucleic acid sequence of the target sequence or a part thereof. In some embodiments, this sequence of the mediator probe is 100%, 99%, 95%, 90%, or 80% complementary to the target sequence. In some embodiments, a mediator probe can tolerate one or more mismatches to the target sequence and still bind to it. In other embodiments, the mediator probe only binds to a target sequence if it matches the target sequence 100%.

[0094] The term "nucleic acid amplification reaction" refers to any method that involves an enzymatic reaction that enables the amplification of nucleic acids. A preferred embodiment of the invention relates to a polymerase chain reaction (PCR). The polymerase chain reaction ("PCR") is the gold standard method for rapidly producing millions to billions of copies (full or partial copies) of a given DNA sample, thereby enabling the amplification of a very small DNA sample to a sufficiently large amount. PCR amplifies a specific region of a DNA strand (the target DNA sequence), depending on where the primers used bind to initiate the amplification reaction. Almost all PCR applications use a heat-stable DNA polymerase enzyme, such as Taq polymerase.Quantitative PCR (“qPCR”) or “real-time PCR” is a specific form of PCR and is a standard method for detecting and quantifying a specific target sequence or for quantifying the gene expression level in a sample in real time. In qPCR, fluorescently or electroactively labeled probes or nucleic acids (e.g., mediator probes) are hybridized in the PCR reaction and, in embodiments, cleaved or digested by the PCR polymerase during primer extension as soon as they bind to a complementary sequence (e.g., a target sequence). In embodiments, the presence and amplification of target sequences is monitored in real time after or during each PCR cycle. This may also include monitoring hybridization and / or extension reactions on a solid phase at a specific time point during a PCR cycle.Real-time PCR allows the progress of an ongoing amplification reaction to be monitored as it occurs (i.e., in real time). Data are therefore collected throughout the entire duration of the PCR reaction, not at the endpoint as with conventional PCR. Measuring reaction kinetics in the early stages of PCR offers distinct advantages over conventional PCR detection. In real-time PCR embodiments, reactions are characterized by the time point during the cycle at which amplification of a target is first detected, rather than by the amount of target accumulated after a fixed number of cycles, as with classical PCR. The higher the starting copy number of the nucleic acid target, the sooner a significant signal change (e.g., fluorescence, electrochemical signal) is observed.Real-time PCR enables analysis using optical signals to detect a specific PCR product (the target sequence), using specific fluorochromes or fluorophores. An increase in DNA product during PCR therefore leads to an increase in the fluorescence intensity measured at each cycle. Using different colored labels, fluorescent probes can be used in multiplex assays to monitor multiple target sequences. For embodiments in which oligonucleotides are immobilized on the solid phase and amplification products interact with them, the position at which the signal change occurs can be used to distinguish between multiple target sequences, in addition to the characteristics of the label (e.g., wavelength of the fluorophore, redox potential of the electroactive molecule).

[0095] While real-time qPCR depends on determining the relative amount of target nucleic acid in each amplification cycle, digital PCR, on the other hand, allows for the determination of the absolute amount of target nucleic acid based on Poisson statistics, which is applied following endpoint PCR amplification to calculate the target nucleic acid amount. The steps prior to amplification are usually comparable or similar between digital PCR and qPCR. However, in qPCR, all nucleic acid molecules are preferably pooled and subsequently amplified and analyzed, whereas in digital PCR, the nucleic acid molecules are preferably distributed as best as possible across individual partitions (e.g.,Emulsion drops, wells, or gel beads), allowing the PCR to run as a single reaction in each partition (in the case of emulsion drops, this reaction is often referred to as droplet PCR or digital droplet PCR) and allowing each partition to be analyzed separately. The random distribution of nucleic acid molecules into individual partitions in digital PCR follows the Poisson distribution. When analyzing the digital PCR, Poisson statistics are then applied to determine the average number of nucleic acid molecules per partition (none, one, or more). The statistical Poisson analysis of the number of positive and negative reactions provides precise absolute quantification of the target sequence.

[0096] The specificity of the mediator probes also prevents interference with measurements by primer dimers, which are undesirable potential byproducts of PCR. In one embodiment, the invention relates to a method wherein the amplification is a multiplex PCR with more than one primer pair. Multiplex PCR is a variant of standard PCR in which two or more target sequences can be amplified and / or detected simultaneously in the same reaction by using at least one primer pair in the reaction.

[0097] In the context of the invention, a "signal change" preferably describes a significant, differentiable and / or characteristic change in the signal, which is clearly demarcated or distinguished from potential baseline or background signals or background or background noise. The person skilled in the art is aware that under some experimental conditions in the context of electrochemical detection, non-specific baseline signals or background or background noise can occur due to non-specific processes at the electrodes, for example non-specific adsorption or aging effects. Therefore, a signal change in the context of the invention preferably describes a significant, differentiable and / or characteristic change in the signal, and not a baseline or background signal or background or background noise. In preferred embodiments, this signal change can be an increase in the current intensity (e.g.Peak currents) and / or the potential and / or the impedance, in other words an increase in the signal. In some embodiments, a signal change is a decrease in the signal. The increase in a signal is preferably due to the fact that an amplification reaction increases the number of target sequence amplifications and thus the activation of associated signal complexes. The more target sequences are present and bound by mediator probes in a PCR reaction, the more or earlier (in the case of a real-time PCR) the signal increases. Preferably, the signal is proportional or approximately proportional to the amount of the corresponding target sequence for which the fluorophore signal (e.g. its color) is specific / characteristic. Since in a digital PCR there is preferably only one target sequence per reaction chamber (e.g. partition, emulsion droplet), the signal increases increasingly with the number of target sequence amplifications per reaction chamber.In preferred embodiments, there is ideally a uniform distribution of a maximum of 1 target sequence per reaction chamber (e.g., partition, emulsion droplet), so that when amplification begins and a similar amplification efficiency is achieved in all reaction chambers (containing a target sequence), the specific signal for detecting an identical target sequence in different reaction chambers is generated with a comparable height / strength / intensity during readout by digital or droplet PCR, which is preferably indicative of the presence and / or number of target sequence amplicons present in each reaction chamber. In embodiments, the intensity / strength of a respective label, preferably specific for a target sequence, as well as the maximum achievable signal strength / intensity can depend on the number of labels per signal oligo and signal complex and / or the type of label (e.g., type of fluorophore and / or quencher).

[0098] “Fluorophore” (or fluorochrome, similar to a chromophore) is a fluorescent chemical compound that can re-emit light when excited by light.

[0099] "Quenching" refers to any process that reduces the fluorescence intensity of a given substance. Quenching ("quenching") is the basis for Förster resonance energy transfer (FRET) assays, static or contact quenching assays, or a combination of both.

[0100] Basically, “surface quenching” refers to the emission-free energy transfer between fluorophores and preferably metallic surfaces.

[0101] In the context of the invention, a “makeshift oligo” refers to an oligonucleotide which, in addition to the signal oligo and the base strand, is required as a third oligonucleotide in order to enhance the signal change by enriching the signal oligo on the solid phase.

[0102] In the context of the invention, "electroactive molecule" preferably refers to a redox molecule that, in the case of electrochemical detection, is reduced or oxidized at defined potentials, whereby electrons are transferred. In this context, "electroactively labeled" preferably means that the electroactive molecule is coupled to an oligonucleotide. Examples of electroactive molecules for labeling oligonucleotides include methylene blue, ferrocene, anthraquinone, etc.

[0103] Implementation examples

[0104] The invention is further described by the following examples. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention, which are provided to illustrate the invention described herein.

[0105] Electrochemical detection

[0106] Unmarked

[0107] To detect an electrical signal change during an amplification reaction, cleavage of the mediator probe and subsequent extension of the mediator on the target sequence-unspecific modular reporter can disrupt the bond of an electroactively labeled signal initiation oligo to the base strand. This oligo can then bind to a temporary oligo immobilized on a solid phase (electrode), thus inducing a signal change (Figure 6 iii). As an alternative approach, the target sequence-independent modular reporter complex can be coupled to a solid phase via a linker on the base strand (Figure 6 i). By extending the mediator, the bond between the signal initiation strand and the electroactive label, or the bond between the electroactive label itself and the base strand, is disrupted, resulting in a signal change.As a further alternative, the signal initiation oligo itself can be immobilized to a surface, and the entire target sequence-unspecific modular reporter complex can be coupled to the electrode via the base strand, which in this case possesses an electroactive modification (Figure 6 ii). By extending the mediator, the bond between the base strand and the signal initiation oligo is broken, resulting in a signal change.

[0108] The probability of hybridization to a solid phase probe can be influenced by the effective free energy of the hybridization reaction and / or by different diffusion rates of molecules of different sizes (length and secondary structure of the DNA (complex)).

[0109] In general, it is conceivable that this approach could be extended to other detection principles, where the signal is detected by enrichment / depletion of signal-generating labels on the solid phase. Among other things, fluorophores could be used for optical detection or magnetic particles for magnetoresistive detection (detecting the movement of ferromagnetic materials via changes in magnetic flux).

[0110] Electroactive labeling on solid phase

[0111] Another option is the immobilization of the electroactive label to the solid phase (electrode), either via the base strand (Figure 6 iv) or the signal initiation oligo (Figure 6 v). In this case, a signal change is achieved by changing the distance from the electroactive molecule to the surface as a result of the severing of the bond between the signal initiation oligo and the base strand. In this case, extending the mediator causes the distance to the surface to either increase (iv) or decrease (v), which in both cases leads to a signal change.

[0112] These examples are not limited to electrochemical detection. Changing the distance of a fluorescent label can lead to a detectable signal change, for example, through surface quenching effects.

[0113] Detection without oligo-modified solid phase

[0114] A further embodiment comprises electrochemical detection comparable to optical detection, as shown, for example, in Figure ?. After hydrolysis of the labeled signal initiation oligonucleotide, the label exhibits a detectably higher diffusion rate and can thus be detected even without enrichment by an oligonucleotide immobilized on the electrode (The effect was described, for example, in Pearce, DM, Shenton, DP, Holden, J., Gaydos, CA, 2011 . IEEE Trans. Biomed. Eng. 58 (3), 755-758. However, in the embodiment described there).

[0115] Results

[0116] To demonstrate a detection reaction following the reporter complex assembly shown in Figure 6 i), a mediator extension reaction was performed in the presence of the electrode surface functionalized with the base strand. The reaction mix comprised a PCR mix (including polymerase, cofactors, etc.) as well as 300 nM of the electroactively labeled molecule (signal oligo), and 200 nM of either the mediator or the intact mediator probe. The sequences of the oligonucleotides are listed in Table 1. The reaction mix was initially incubated for 5 min at 95 °C to activate the polymerase.

[0117] The reaction mix was then transferred into reaction chambers of a functionalized electrode array. The closed reaction chambers were initially incubated in a water bath at 40 °C and then heated to up to 62 °C. During the heating phase and the subsequent cooling process, the electrodes were electrochemically analyzed using square-wave voltammetry. Upon enrichment of the electroactive molecule at the electrode surface through hybridization, the current signal exhibits a characteristic peak (Aip ea k) appears.

[0118] Table 1: Sequences for the mediator extension reaction according to Figure 6 i). Sequence segments belonging to the receptor complex are shown in italics. Sequence segments belonging to the signaling complex are underlined.

[0119] The result (Figures 2 and 3) initially shows clearly detectable electrochemical signals at temperatures below 45 °C, which, however, do not differ between the different reaction mixes. This indicates hybridization of the signal oligo to the solid-phase probe, but no significant displacement reaction of an extended mediator. With increasing temperature and thus increasing polymerase activity, the signal for reactions containing mediators begins to decrease and differ from reactions with mediator probes. This indicates that the mediator on the solid-phase oligo is extended by polymerase activity, thereby displacing the electroactively labeled oligonucleotide. Even after or during the cooling process, the signals from reactions with active mediator remain distinguishable from those with mediator probes (Figure 3).

[0120] For real-time PCR, the active mediator must be generated by cleavage of the mediator probe. In this case, in addition to polymerase, PCR buffer, and 200 nM mediator probe, target sequence-specific primers (> 200 nM) are added to the PCR mix. For positive controls (PTC), a target DNA containing the target sequence to be amplified is added, while the "no-template control" (NTC) does not contain any target DNA. The base strand is immobilized on the surface of the working electrode. The signal oligo can initially be pre-hybridized with the base strand or be part of the PCR mix. For real-time detection, the PCR takes place in a chamber containing the functionalized electrode. For end-point PCR detection, the amplification reaction can also take place in an upstream chamber. The PCR is carried out according to the recommended temperature protocol for the polymerase used, i.e.after activation of the hot-start polymerase by cyclic temperature changes between 95°C and approximately 60°C. The signal is detected during the approximately 60°C temperature step (typical temperature for the annealing of primers and probes to target sequences). Cleavage of the mediator probe during the PCR generates increasingly activated mediators, which hybridize to the base strand and, through elongation, lead to a detectable displacement of the electroactively labeled signal oligo or, in the next temperature step, prevent its rehybridization to the base strand. This causes a decrease in signal; in endpoint detection, this may be compared to NTC reactions that use uncleaved mediator probes. A prerequisite for real-time detection is that the melting temperature (Tm) of the mediator and signal oligo at their respective binding sites is above the evaluation temperature.With endpoint detection, there is greater flexibility regarding the melting temperature, as mediator extension and signal oligo hybridization can take place below the primer annealing temperature. Previous results indicate that sufficient polymerase activity for the mediator extension and signal oligo displacement reaction is present up to a temperature of 45 °C, meaning an extended temperature window between 45 °C and approximately 60 °C (and even higher with appropriately designed Tm of mediator and signal oligo) is possible.

[0121] For a detection reaction following the reporter complex assembly shown in Figure 6 ii), a mediator extension reaction was performed under PCR conditions in a standard PCR cycler. The electroactively labeled molecule (base strand) and the mediator or the intact mediator probe as a control were added to the PCR reagents. After 10 cycles, the reaction result was transferred to an electrode array functionalized with the signal oligo. The hybridization reaction taking place there at room temperature was analyzed electrochemically using square-wave voltammetry. The sequences used are listed in Table 2.

[0122] The result (Figure 4) shows an increasing electrochemical signal for reactions containing an intact mediator probe (dark gray (upper) curve). Surprisingly, no electrochemical signals are detectable for reactions containing mediators (light gray (lower) curve). This indicates an effective extension reaction of the mediator on the electroactively labeled strand, which subsequently leads to an effective prevention of the hybridization reaction on the functionalized electrode surface. An elevated temperature or polymerase activity is no longer required for the detection step; however, the hybridization rate would increase at a higher hybridization temperature, and the signal plateau would be reached more quickly. Table 2: Sequences for the mediator extension reaction according to Figure 6 i). Sequence segments belonging to the receptor complex are shown in italics.Sequence segments belonging to the signaling complex are underlined.

[0123] For a real PCR, the addition of target sequence-specific primers to the previously mentioned reaction components (polymerase, buffer, electroactively labeled base strand) is also necessary in this case, as is the target DNA for the PTC. During the procedure, PTC and NTC reactions are set up and PCR cycles (between 95°C and approximately 60°C) are carried out. For real-time detection, the electrode functionalized with a signal oligo is in the reaction chamber. For end-point detection, this is not mandatory. In the PTC, mediator probes are cleaved during the primer extension reaction during the PCR. The activated mediator hybridizes to the base strand and is extended there, separating the base strand from the immobilized signal oligo and / or preventing its rehybridization to the signal oligo. Signal detection in real-time PCR occurs at the end of each primer / mediator extension step at approximately 60°C.A decrease in signal is observed with PTC. As previously mentioned, the melting temperatures of the signal complex must be close to or above the analysis temperature. In the case of endpoint detection, the signal from PTC is lower than that from NTC reactions. The analysis temperature can be selected from room temperature to the melting temperature of the signal complex, with a higher temperature being preferable due to the faster hybridization rate and the avoidance of nonspecific hybridization processes.

[0124] Figures

[0125] Figure 1: The figure shows the Mediator probe cleavage during PCR with Mediator activation in one embodiment of the invention. The "activated" Mediator here corresponds to a Mediator sequence that is released after digestion of the Mediator probe.

[0126] Figure 2: Electrochemical signal as a function of time for the execution of a mediator extension reaction according to the embodiment of Figure 6 i) in the presence of the mediator (active) or the mediator probe. The peak currents (A / p e ak) were normalized to time t = 5 min. The temperature shown refers to the temperature of the water bath.

[0127] Figure 3 Quotient of normalized electrochemical signal of reactions with mediator to

[0128] Reactions with a mediator probe (see Figure 2) as a function of the water bath temperature. The plot distinguishes between signals recorded during the heating phase (squares) and the cooling phase (crosses).

[0129] Comparison of the electrochemical signal of a hybridization reaction after thermocycling in the presence of electroactively labeled oligonucleotides and mediators (light gray) or intact mediator probes (dark gray) according to Figure 6 ii).

[0130] The figure shows an example of electrochemical signal generation according to

[0131] Mediator activation using a target sequence-nonspecific modular reporter complex.

[0132] The figure shows an example of electrochemical signal generation according to

[0133] Mediator activation using a target sequence-unspecific modular reporter complex. As one embodiment, the target sequence-independent modular reporter complex can be coupled to a solid phase via a linker on the base strand (Figure 6i). By extending the mediator, the binding of the signal initiation strand with the electroactive label or the binding of the electroactive label itself to the base strand is disrupted, resulting in a signal change. As a further embodiment, the signal initiation oligo itself can be immobilized to a surface, and the entire target sequence-unspecific modular reporter complex can be coupled to the electrode via the base strand, which in this case has an electroactive modification (Figure 6ii).As a further embodiment, a signal initiation oligo can bind to a makeshift oligo immobilized on a solid phase (electrode), resulting in a signal change (Figure 6iii). Two further embodiments are the immobilization of the electroactive label to the solid phase (electrode), either via the base strand (Figure 6iv) or the signal initiation oligo (Figure 6v). In this case, a signal change is achieved by changing the distance from the electroactive molecule to the surface as a result of the severing of the bond between the signal initiation oligo and the base strand. In this case, extending the mediator causes the distance to the surface to either increase (Figure 6iv) or decrease (Figure 6v), which in both cases leads to a signal change.

[0134] Figure 7 The figure shows a further embodiment, wherein the detection is carried out without immobilized

[0135] After hydrolysis of the labeled signal initiation oligonucleotide, the label exhibits a detectably higher diffusion rate and can thus be detected without enrichment by an oligonucleotide immobilized on the electrode.

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Claims

Patent claims 1. Method for detecting at least one target nucleic acid sequence, comprising the steps of: a. providing at least one target sequence-nonspecific modular reporter complex, comprising at least one label, and at least two oligonucleotides, namely i. a basic strand, comprising 1. at least one mediator liaison point 2. at least one signal initiation oligo binding site; ii. at least one signal initiation oligo; b. providing at least one mediator probe, wherein the mediator probe comprises an oligonucleotide with at least one probe sequence and at least one mediator sequence, wherein the at least one probe sequence has an affinity for at least one target nucleic acid sequence, and the at least one mediator sequence has an affinity for at least one mediator binding site on the base strand of the at least one target sequence-nonspecific modular reporter complex; c. PCR amplification of at least one nucleic acid sequence; d. binding of a probe sequence of at least one mediator probe to at least one target nucleic acid sequence; e. cleavage of the probe sequence of the at least one mediator probe bound to the at least one target nucleic acid sequence by a PCR polymerase, wherein the mediator sequence is released; f.g. Binding of at least one released mediator sequence to a mediator binding site of the at least one target sequence-nonspecific modular reporter complex, g. Extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, wherein the binding (hybridization) of the at least one signal initiation oligo binding site and the at least one signal initiation oligo is either i) disrupted and / or prevented or ii) initiated and / or established, thereby initiating a signal change, h. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence.

2. Method according to claim 1, wherein in step g. a signal change is initiated by the separated at least one signal initiation oligo comprising at least one marker and binding to at least one auxiliary oligo which is immobilized on a solid phase. A method according to claim 1, wherein in step g. a signal change is initiated by immobilizing one or both oligonucleotides of the at least one target-sequence-unspecific modular reporter complex, namely i. the base strand, and / or ii. the at least one signal initiation oligo, via a linker on a solid phase. A method according to claim 3, wherein either the base strand or the at least one signal initiation oligo comprises the at least one label. A method according to claim 4, wherein the signal change in step g. is based on the fact that the extension of the mediator sequence leads to an increased or decreased hybridization probability of the labeled oligonucleotide on the immobilized oligonucleotide, thereby enriching or decreasing the label on the functionalized surface.A method according to any one of the preceding claims, wherein the at least one marker comprises at least one electroactive marker and the solid phase is an electrode. A method according to claim 6, wherein in step g. a signal change is initiated by increasing or decreasing the distance between the at least one marker and the solid phase in step g. A method according to claim 6, wherein the signal change in step g. is based on the fact that the number of bound markers results in a change in the electrode potential or the charge transfer. A method according to claims 7 and 8, wherein the described change is determined as a change in current or charge or potential or charge transfer resistance by means of, for example, voltammetric, amperometric, coulometric, potentiometric or impedimetric measurement methods. A method according to claims 1 to 5, wherein the at least one marker comprises at least one fluorophore.A method according to claim 10, wherein the signal change is based on the fact that the number of bound labels results in a change in the fluorescence intensity. A method according to claim 10, wherein the signal change is based on the fact that a change in the distance of the label to the solid phase causes a signal change. The solid phase is a (preferably metallic) surface that causes fluorescence quenching of proximal fluorophores. A method according to claims 1 to 5, in which the at least one label is a molecule for coupling further labels. A method according to claim 13, wherein an optically detectable marker such as colloidal gold, colloidal silver, colloidal carbon, or latex particles is coupled to the marking. A method according to claim 14, wherein the signal change is an optically perceptible change (for example, by eye or camera) in the color intensity at the location of the immobilized oligonucleotides. A method according to claim 13, wherein a nanoparticle (preferably a metal nanoparticle) is coupled to the marking and the solid phase is a piezoelectric material that is excited to vibrate by an alternating electric field. A method according to claim 16, wherein the signal change in step g is a change in the frequency and / or phase and / or damping of the vibration depending on the number of bound markings. A method according to claim 13, wherein a metallic nanoparticle is coupled to the marking and the solid phase is a metallic surface.A method according to claim 18, wherein the signal change is based on a change in the refractive index at the surface due to the increase / decrease of bound labels, which can be determined by surface plasmon resonance spectroscopy. A method according to claim 13, wherein magnetic nanoparticles are coupled to the label and the signal change is based on a change in the resistance of a magnetoresistive sensor. A method according to any one of the preceding claims, wherein the immobilized oligonucleotides can be immobilized at several defined positions in the solid phase. The immobilized oligonucleotides can differ in their sequence. A method according to any one of the preceding claims, wherein the target sequence is uniquely identifiable via the characteristics of the label and / or the position of the signal change.A method according to claim 1, wherein the separated at least one signal initiation oligo comprises at least one electroactive label, and wherein in step g. the at least one electroactive label is released, thereby exhibiting a detectably higher diffusion rate and thus initiating a signal change, preferably by increasing the electron transfer rate. A method according to any one of the preceding claims, wherein the at least one label is specific for the at least one target nucleic acid sequence, and wherein the signal change initiated in step g. is characteristic of the at least one target nucleic acid sequence. A method according to any one of the preceding claims, wherein the detection of the signal change comprises an analysis of the signal change as a function of the detection temperature and / or the electric field and / or the frequency of the excitation signal. A method according to any one of the preceding claims, wherein in step a) the signal initiation oligo binding site of the base strand and the at least one signal initiation oligo are hybridized but not covalently linked and together form a signal complex. A kit for carrying out the method according to any one of the preceding claims comprising: at least one oligonucleotide primer, at least one mediator probe, at least one signal initiation oligo, at least one base strand, and at least one buffer. PCR polymerase.

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