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

Abstract

The invention relates to a method for detecting at least one target nucleic acid sequence by means of a mediator probe and at least one target-sequence-unspecific modular reporter complex, wherein the released mediator sequence binds to a mediator binding site of the target-sequence-unspecific modular reporter complex, and is extended. A signal change is initiated and detected. The invention also relates to a kit for carrying out this method.

Description

[0001] Nucleic acid detection in a PCR using a target-unspecific modular reporter complex

[0002] Technical background

[0003] In quantitative detection reactions of PCR products such as real-time PCR (qPCR) or digital PCR (dPCR), target sequences are 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 through cleavage either directly (e.g. TaqMan probes) or indirectly (mediator probes in combination with universal reporter molecules). 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).

[0004] 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. However, these probes are easy to synthesize, as usually only two terminal labels are required. In addition to these target sequence-specific DNA probes, there are also fluorogenic detection molecules that are target sequence-nonspecific (e.g. mediator probes and universal reporters).These have the disadvantage that their synthesis is significantly more complex, but they enable entirely new types of sequence detection and feature highly optimized fluorescence signal generation. In addition to these types of single-part detection molecules, there are also two-part, target sequence-specific detection probe systems (light-cycler probes). In these systems, a fluorescence signal between two neighboring DNA probes is shifted into the longer-wavelength spectral range via their fluorophores using FRET, provided both bind correctly to the DNA target sequence, thus establishing the required separation. Such systems also suffer from the problem of sequence specificity and require extremely fine-tuned design.

[0005] 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). When the ends are annealed to form a loop due to the complementary bases, the fluorescent molecules are brought into close proximity. This transfers the fluorescence from the donor to the acceptor, whereby the emitted light wavelength is changed or quenched by energy loss to the longer wavelength of the acceptor. Only after the oligo binds to the complementary region of the target sequence and the concomitant opening of the loop is the fluorescence quenching reversed, generating an increase in the fluorescence signal. This enables the detection of the light energy of the fluorescent donor.

[0006] TaqMan probes also consist of an oligonucleotide and two terminal fluorescent molecules (Heid et al. 1996). However, the oligonucleotide does not form a loop; instead, the fluorescence is transferred through spatial proximity on the oligonucleotide. Upon annealing of the probe to the target gene and extension of a primer, the probe is degraded by the exonuclease activity of the polymerase used, thus separating the fluorophore from the quencher. If fluorescence quenching is insufficient due to a particularly long probe sequence and the resulting large spatial separation of the fluorescent molecules, an additional internal or terminal quencher can be added. However, this is more challenging because the exact length of the cleaved sequences is often unknown, and it is therefore possible that the cleaved sequence with the fluorescence donor also contains one of the internal quenchers.In digital PCR, Taqman probe systems are also used for so-called "intensity multiplexing." This involves using Taqman probes with identical fluorescent labels but different DNA sequences to differentiate multiple target sequences in a multiplex PCR. This is made possible by using different types of Taqman probes at different concentrations. However, this method also requires complex fine-tuning of the concentrations and is usually not very precise (Whale et al. 2016). The modular reporter complex described in this invention has the advantage that different fluorescence intensities can be adjusted at constant concentrations of detection molecule complexes by forming complexes of multiple fluorophore- and quencher-labeled oligonucleotides. This requires less fine-tuning and enables more precise signal adjustment.In addition to direct optical signal generation using a fluorescently labeled DNA probe, there are also systems where detection occurs via a DNA probe that does not carry a fluorescent label. These also bind specifically to the target sequence and are cleaved by the exonuclease activity of a polymerase. In contrast to the probe types described so far, this cleavage does not generate a fluorescent signal; instead, signal generation is initiated by a second molecule, which is itself independent of the target sequence. This has already been demonstrated in both PCR and LAMP (Faltin et al. 2012; Faltin et al. 2013).

[0007] One of these methods is called mediator probe PCR, for which a patent was filed by the University of Freiburg in 2012. In this system, two oligonucleotides are used in the PCR for detection instead of just one. These are referred to as the mediator probe (MP) and the universal reporter (UR). During PCR amplification of the target sequence, only a portion of the mediator probe binds in a sequence-specific manner and is cleaved by the exonuclease activity of the polymerase. In this process, a second portion of the mediator probe, the mediator, which was not previously bound to the sequence, is separated. If no target sequence is present, the mediator probe remains intact. After separation of the mediator, it will in turn bind to a universal reporter.A universal reporter is target sequence-independent and, in addition to the mediator binding site, also possesses a fluorophore and quencher modification, as well as a conformation that brings both modifications into spatial proximity. By extending the mediator on the universal reporter, this spatial proximity is eliminated, e.g., by cleaving the fluorophore, resulting in a fluorescence signal of a specific wavelength. This procedure has the advantage of separating probe binding to the DNA sequence from fluorescence signal generation. This allows for clear guidelines for mediator probe design, allows universal reporters to be extensively optimized once and then used for multiple sequences, and, moreover, this two-part process provides double control, making signal generation highly specific (Lehnert et al. 2018; Wadle et al. 2016, Schlenker et al. 2021).A disadvantage of this technology, however, is that corresponding universal reporters are very complex and expensive to synthesize, as some of these modifications must occur internally within the DNA sequence. This makes further development of these molecules to impart new properties very complex and difficult. Furthermore, additional protecting groups are required on a universal reporter.

[0008] A further disadvantage of this technology is that the fluorescence ranges of a color channel differ depending on the device, requiring a separate fluorophore-quencher optimization for each device. This optimization is very complex and cost-intensive, as a new universal reporter must be synthesized for each combination. Furthermore, the structure of the oligonucleotide is limited, so there are only a few options for attaching additional labels for stronger signals or multiplex variations. This severely limits the flexibility of the reporter with regard to testing different labels. The disadvantage of this system is the very expensive production of the universal reporter due to its multiple modifications, as well as its inflexible design. During the same period, a technology from Seegene Inc. was developed., which is also based on the separation of DNA sequence detection and signal generation via two detection molecules in real-time PCR. An unlabeled PTO probe binds to a DNA target sequence, is cleaved, and releases a fragment, which then forms an extended duplex with a fluorescently labeled target sequence-nonspecific detection molecule (CTO molecule). This process is used to influence signal generation across different lengths of these detection molecules. This enables, for example, the differentiation of multiple target sequences in the same detection channel by reading the signal at defined, pre-specified temperatures. Similar to a universal reporter, a CTO is also a single molecule that places corresponding demands on its synthesis.

[0009] Another patent also uses readout at different temperatures in a single approach and can therefore be interpreted as a further development of the aforementioned Seegene technology (PCT / CN2018 / 084794). Here, too, an extended reporter molecule is melted after detection. In contrast to the previous patent, a single reporter molecule can be used to differentiate between different target sequences.

[0010] Patent W02013079307A1, "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 universal reporter (UR) (mediator hybridization sequence). After cleavage, the mediator probe acts as a primer. This extends the primer and separates the fluorophore and quencher at the universal reporter.

[0011] Patent WO2018114674A1 on the loop-mediated isothermal amplification method with mediator-displacement probes (MD LAMP) claims a universal reporter with at least one oligonucleotide and at least one fluorophore and quencher for a LAMP reaction. Previously, it was assumed that this only works because, unlike PCR, LAMP continuously maintains a uniform temperature, allowing equilibrium to be quickly established and maintained, allowing individual molecules to bind permanently to one another and thus not generate a signal in the initial state.

[0012] In the current state of science and technology, optical detection reactions use different wavelengths to distinguish individual target sequences within specific light ranges, known as 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.

[0013] 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.

[0014] The modular reporter complex described in this invention has the advantage that, in certain embodiments, multiple target sequences can be detected in a single detection channel independently of temperature. This is achieved by using multiple fluorophores or quenchers on a signal initiation or base strand, which provide different fluorescence intensities and thus make fluorescence signals assignable within the same channel. This allows detected target sequences to be differentiated based on the fluorescence intensity generated. This enables the simultaneous detection of at least two target sequences within a single detection channel without complex temperature-dependent readout steps.

[0015] Another patent is the US-only patent from Biorad (US 9921154 B2). This also claims the detection of multiple target sequences in identical detection channels in a digital PCR, but only describes sequence-specific hydrolysis probes labeled with fluorophore and quencher or intercalating dyes for such detection. Other patents from Biorad also describe the differentiation of different target sequences, sometimes in a single channel. However, according to current understanding, these can only be used effectively if there is a significant overcrowding of the droplets.

[0016] Object of the invention

[0017] PCR is the gold standard method for amplifying individual DNA sequences and thus making them detectable. To detect and quantify PCR products (DNA or cDNA in the case of RNA), either intercalating dyes or DNA probes are used. 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] With optical detection, e.g. via fluorescence, such detection molecules or detection molecule systems also have the disadvantage that they usually have to have multiple biochemical labels and modifications at once (e.g. fluorophore and quencher in the case of a fluorogenic Taqman probe, fluorophore, quencher and 3'-block group in the case of a target sequence-nonspecific universal reporter). This makes them more complex and expensive to synthesize and thus inflexible in their design. This is a major problem, particularly in the development and optimization of DNA detection reactions, as only a small number of detection molecule systems can be tested. Furthermore, the possibilities of detection molecules and thus detection methods are significantly limited, as an unlimited number of modifications can not be made to a single DNA sequence.However, since the bonds between DNA sequences are broken and reconstituted multiple times during PCR, the current state of the art assumes that all biochemical modifications must always be bound to a DNA sequence in a fluorogenic DNA detection molecule. This is the current assumption in the state of the art that is the only way to ensure the initially required spatial proximity between fluorophore and quencher in order to suppress the fluorophore's signal by the quencher until these molecules are spatially separated during the detection process. Furthermore, the molecular processes that lead to such signals are not always uniform, as probes can, for example, either be cleaved or unfolded to generate signals. This, however, leads to inhomogeneous signal generation, which leads to less precise results.

[0019] This highlights the urgent need for a new type of detection processes and signaling molecules that overcome these problems.

[0020] The inventors determined that this task could be solved by 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 described by the current state of the art for single-component detection molecules. 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, as the detection molecules themselves would be separated during thermal cyclic heating.

[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 oligo binding site ii. at least one signal oligo wherein the at least one signal oligo binding site of the base strand and the at least one signal oligo hybridize 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 the 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 with nuclease activity during PCR amplification, 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 oligo binding site and the at least one signal oligo is broken, thereby initiating a signal change, h. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence.

[0026] In a preferred embodiment of the method according to the invention, the at least one label of the target sequence-nonspecific modular reporter complex comprises at least one fluorophore and / or at least one quencher.

[0027] In embodiments, the present method serves to detect at least one nucleic acid sequence during a PCR reaction, wherein a mediator probe is cleaved during this reaction (by an exonuclease activity of the polymerase). The cleavage product is a mediator, which subsequently binds to a target sequence-nonspecific modular reporter complex and, via a subsequent reaction, initiates a signal change that serves to detect the DNA sequence. In this embodiment, the target sequence-nonspecific modular reporter complex preferably consists of at least two oligonucleotides that are not covalently linked, wherein at least one oligonucleotide of this complex has at least one label that initiates a signal change and which preferably comprises at least one fluorophore and at least one quencher.In this embodiment, an oligonucleotide of the target sequence-nonspecific modular reporter complex (base strand) preferably has at least one binding site for at least one mediator sequence (also referred to herein as a receptor) and at least one binding site for a signal oligo. In this embodiment, the second oligonucleotide of the complex is the signal oligo, which binds to the base strand (thus forming a signal complex). As long as the mediator probe is uncleaved, the target sequence-nonspecific modular reporter complex remains in its ground state during the detection or readout process.By extending the mediator oligonucleotide along the base strand during PCR, the target sequence-unspecific modular reporter complex at the signal complex is disrupted in such a way that the label or labels on the base strand and / or on the signal oligo are separated from each other, thereby initiating a change of the signal to the ground state and thus a signal change, which serves to detect the DNA target sequence.

[0028] In some embodiments, steps d-h of the method according to the invention can be carried out continuously in each cycle during the PCR amplification. This is preferably the case when the PCR amplification is a real-time PCR or qPCR. In other words, in embodiments of the method according to the invention, steps d-h are repeated in each PCR cycle during the PCR amplification, wherein the PCR amplification is a real-time PCR or qPCR.

[0029] In other embodiments, steps d-g of the method according to the invention can take place during the PCR amplification, with step h following subsequently; this is preferably the case when the PCR amplification is a digital PCR or endpoint analysis. In other words, in embodiments of the method according to the invention, steps d-g are repeated in each PCR cycle during the PCR amplification, and step h follows subsequently, with the PCR amplification being a digital PCR or endpoint analysis. In the case of a digital PCR and / or endpoint analysis, the detection step h of the method according to the invention preferably takes place separately, following the PCR amplification reaction.

[0030] The modular reporter system for target sequence-nonspecific detection according to the invention differs from previously known detection systems through the flexible use of various oligonucleotides with different labels. Surprisingly, it exhibits the same performance characteristics in a PCR as current single-part detection molecules. Above all, the modular design offers various advantages, such as the flexibly adaptable design 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 a PCR.The core of the invention is the modular, target-sequence-independent modular reporter complex (see Figure 2A for an exemplary embodiment) composed of non-covalently linked oligonucleotides. This system possesses all the advantages of various target-sequence-specific detection systems as well as target-sequence-nonspecific detection molecules and combines them with increased flexibility in the design of such detection molecules, leading to entirely new detection methods. Surprisingly, the system can be used without problems in a PCR with cyclic temperature changes and the associated melting of the DNA strands in each cycle. The results of the present examples, which are also shown in Figures 6-8, provide evidence that the modular system according to the invention even surpasses the functionality of prior art universal reporters (UR).This allows for significantly lower production costs while maintaining at least equally good performance parameters. A further advantage of the system is the ability to flexibly adapt the universal probe according to the invention. Different fluorophores and quenchers can be combined and investigated with one another, and multiplex reactions can be expanded. Using different labels, different probes can be differentiated in a single fluorescence channel. Thus, the invention provides a dynamic, cost-effective, and universal detection system.

[0031] 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 preferably has at least one binding site for a mediator (receptor complex) and at least one binding site for a signal initiation oligonucleotide (or “signal oligo” for short) and thus forms a signal complex (examples of embodiments comprising a signal complex and a receptor complex can be found, for example, in Figures 2, 4 and 5). The base strand and at least one signal oligo together form a complex (see, for example, Figure 2B for an exemplary embodiment). Preferably, at least one of these strands has a label which can detect a structural change in this complex. This structural change preferably represents the separation, displacement orThe cleavage, detachment, or enzymatic digestion of the signal oligonucleotide from the base strand preferably leads to a signal change. The modular design results in embodiments from several possible signal initiation oligonucleotides (or signal oligonucleotides, "signal oligos" for short) as well as additional labels, which can be attached to both the signal oligonucleotides and the base strand (see, for example, Figure 2B, label positions Li to Li on the signal complex). Various options for attaching cis- or trans-labels allow the advantages of state-of-the-art target gene-dependent systems to be utilized. Various exemplary embodiments can be found in Figures 4 and 5. The modular design allows a much higher number of labels to be attached to each complex than is possible with current single-part detection molecules.

[0032] Thus, in embodiments of the method according to the invention, the base strand comprises at least one signal oligo binding site to which two or more signal oligos are hybridized, and wherein the two or more signal oligos and / or the base strand have one or more labels at the at least one signal oligo binding site. Due to this novelty of the modular detection complex, new possibilities arise for detection reactions based on a basic reaction, which is described below using the example of optical detection (see Figures 1 and 2A for an exemplary embodiment):

[0033] In one embodiment, a Mediator probe binds to the amplified DNA target sequence during a PCR detection reaction. A Mediator probe is preferably an oligonucleotide and has a sequence-specific probe segment that binds to the target sequence and is protected at the 3'-terminal nucleotide, and a target-sequence-nonspecific segment, called the Mediator, which does not bind to the target sequence except for one nucleotide shared by the Mediator and probe. During primer extension by a polymerase with exonuclease activity (e.g., during an amplification reaction), the Mediator is cleaved from the probe, leaving the shared 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 (Figure 2A).

[0034] In some preferred embodiments, a mediator probe therefore 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.

[0035] 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.

[0036] In embodiments, a Mediator probe protected at the 3' end may comprise a "C3 spacer." Such a C3 spacer may be a chemical block group, which in some embodiments comprises a chain of three carbon atoms. This "C3 spacer" thus preferably prevents (nonspecific) polymerase extension of the Mediator probe sequence strand. Typical block groups (protecting groups) suitable for specific embodiments 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 block groups (protecting groups) as routine adaptations of the invention described herein.

[0037] In preferred embodiments of the method according to the invention, no fluorescence signal change is generated by the at least one fluorophore if the at least one signal oligo is hybridized with the at least one signal oligo binding site of the base strand, wherein either the at least one quencher is localized at the at least one signal oligo binding site of the base strand and the at least one fluorophore is localized at the at least one signal oligo or vice versa, and wherein in step g at least one fluorophore and at least one quencher are separated, thereby initiating a signal change.

[0038] In the context of the invention, a fluorescence signal change preferably describes a significant, differentiable, and / or characteristic change in the fluorescence signal that is clearly distinct from or differentiated from potential baseline or background signals or background or background noise. Therefore, in the context of the invention, a fluorescence signal change preferably describes a significant, differentiable, and / or characteristic change in the fluorescence signal, and not a fluorescence baseline or background signal or background or background noise.

[0039] In embodiments, the at least one label further comprises at least one fluorophore and at least one quencher, wherein both the at least one quencher and the at least one fluorophore are localized on the at least one signal oligo, and wherein in step g. (extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase), the at least one signal oligo is cleaved by the PCR polymerase, thereby separating the at least one fluorophore and the at least one quencher, thereby initiating a signal change. In embodiments, the cleavage of the signal oligo from the signal oligo binding site of the base strand by a PCR polymerase can occur either by enzymatic digestion or cleavage of the signal oligo by the polymerase (e.g., by exonuclease activity of the polymerase) or by another mechanism, e.g.,by the signal oligo being detached, separated or displaced from the base strand by the polymerase.

[0040] The inventive process for detecting DNA sequences using PCR in combination with optical readout comprises a novel system of individual oligonucleotides, preferably 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-unit target-sequence-dependent DNA probes or single-unit target-sequence-independent detection molecules.

[0041] The target-sequence-independent modular reporter complex preferably consists of at least two DNA sequences that bind specifically to each other and carry chemical modifications that initiate signal generation during a PCR reaction (e.g., DNA amplification). In some embodiments, the use of different target-sequence-independent reporter complexes with a different number of labels and / or different numbers of signal oligos can generate signals of varying strength, making the activation of these target-sequence-independent reporter complexes distinguishable. Thus, the combination of multiple or different labels (e.g., different colors and / or intensities) can generate distinguishable signals.For example, the signal of a signal oligo with one red marker can be differentiated by color from the signal of a signal oligo with two or three red markers based on the intensity differences of the generated signal, or from the signal of a signal oligo with one red and one green marker. Within the scope of embodiments of the present invention, not only individual markers but also different combinations of different fluorophore colors and / or the number of fluorophores (signal intensity) are provided, each of which encodes a signal specific for a target sequence. This combinability of signals is advantageous for the detection of multiple target sequences at the same time (in the same PCR reaction).

[0042] Multiplex detection reactions, i.e., the simultaneous detection of different target sequences in a sample and in a reaction, require, in the current state of the art, either the availability of multiple optical channels, additional processing steps, or complex concentration adjustment of the reporter molecules. In contrast, the target sequence-independent modular reporter complex according to the invention enables combined detection via different channels, for which a base strand with more than one receptor complex can be used. In these embodiments, several receptor complexes (> 2) are arranged offset along the base strand so that they can activate different signaling complexes (see Figure 3 for an exemplary embodiment). In various embodiments, a receptor complex can regulate or activate one or more signaling complexes, preferably located upstream (towards the 5' end).

[0043] Thus, in embodiments of the method according to the invention, the base strand comprises at least one signal oligo binding site to which two or more signal oligos are hybridized, and wherein the two or more signal oligos and / or the base strand have one or more labels at the at least one signal oligo binding site.

[0044] In some embodiments, the base strand comprises two or more signal oligo binding sites, and wherein at least one of the signal oligos hybridized to the two or more signal oligo binding sites and / or the base strand has one or more labels at at least one of the two or more signal oligo binding sites.

[0045] In some embodiments, the at least one target sequence-non-specific modular reporter complex enables the detection of at least a first and a second target nucleic acid sequence in that the base strand comprises at least a first and a second mediator binding site for at least a first and a second mediator sequence of at least a first and a second mediator probe, as well as at least a first and a second signal oligo binding site to which at least a first and a second signal oligo is hybridized, and wherein the first mediator probe has a probe sequence with an affinity for a first target nucleic acid sequence and the second mediator probe has a probe sequence with an affinity for a second target nucleic acid sequence.

[0046] In some of these embodiments, the base strand comprises at least a first and a second label, wherein the signal change by the at least one first label is characteristic of the first target nucleic acid sequence, and the signal change by the at least one second label is characteristic of the second target nucleic acid sequence.

[0047] In some embodiments, at least a first and a second target sequence-non-specific modular reporter complex are provided in step a., wherein the at least first target sequence-non-specific modular reporter complex enables the detection of at least one first target nucleic acid sequence and the at least second target sequence-non-specific modular reporter complex enables the detection of a second target nucleic acid sequence, wherein the signal change due to the at least one label of the at least first target sequence-non-specific modular reporter complex is characteristic of the first target nucleic acid sequence and the signal change due to the at least one label of the at least second target sequence-non-specific modular reporter complex is characteristic of the second target nucleic acid sequence.

[0048] Preferably, different extended mediator sequences can be encoded using different color combinations, allowing for the detection of different target sequences. Thus, a target sequence-independent modular reporter complex according to the invention is flexible in design.

[0049] Therefore, in some embodiments, the signal changes characteristic of the at least first and the at least second target nucleic acid sequence differ from each other by their color and / or their fluorescence or signal intensity.

[0050] In embodiments, different encodings of fluorescent signals can be used. In one embodiment, a signal complex comprises at least two signal oligos with two labels, where the common signal is characteristic of a target sequence.

[0051] Thus, in embodiments, the combination of several signal oligogos, each with at least one marker, is possible in order to generate a signal specific for a common target sequence. By combining several markers of the same or different colors, signals can be generated which differ from other signals for other target sequences in terms of their color and / or intensity. By combining different fluorescence colors of markers and optionally also in their number, mixed colors can also be generated. In another embodiment, the base strand comprises at least two signal complexes with different signal oligogos and / or different markers on the signal oligogos, wherein the base strand comprises a single receptor complex corresponding to the at least two signal complexes and having at least one mediator binding site, e.g. downstream (towards the 3' end) of the signal complexes.In this embodiment, a target sequence can be encoded by two colors.

[0052] The effect achieved by these embodiments is colorimetrically encoded multiplexing in the digital PCR or qPCR detection or amplification reaction, respectively, which enables the detection of more (a greater number of) target sequences than the number of available detection channels. An example of color coding of fluorophores would be the signal combinations: Target sequence 1: red-red, Target sequence 2: red-green, Target sequence 3: green-green, Target sequence 4: green-green-red.

[0053] The detection of multiple target sequences within a wavelength range can be achieved by quantitatively examining the fluorescence values ​​or in combination with other parameters (e.g., the readout temperature). Signal generation can be influenced by varying the length of the signal oligos, allowing differentiation of multiple target sequences in the same detection channel. For this purpose, specific readout temperatures for signal detection must be defined prior to analysis. Depending on the selected parameters, such as the length of the signal oligo and / or the selected fluorophores, a different signal results at different temperatures. By iteratively reading out the fluorescence signal at different temperatures, a specific signal change can be detected at certain temperature ranges, which is characteristic of the selected fluorescence modifications or lengths of the signal oligos.This allows us to determine which modular reporter complex was activated.

[0054] Therefore, in some embodiments, the detection of the signal change comprises an analysis of the signal change as a function of the detection temperature.

[0055] The inventive method described here has the advantage over the multiplexing method described in US 9921154 B2 that the inventive method can also be used without multiple occupancy of each reaction chamber and / or a set of different types of modular reporter complexes can generate fluorescence signals of varying strengths through multiple labeling via, for example, single or multiple signal oligos that are very easy to apply in this case, without the need for complex synthesis. Without the use of multiply labeled target sequence-specific hydrolysis probes that are complex to synthesize, as described in the method of US 9921154 B2, this method can, according to current understanding, only be used if there is a significant multiple occupancy of DNA target sequences in each reaction chamber of a digital PCR, which thus leads to distinguishable signal clusters in the data space.

[0056] Furthermore, a target sequence-independent modular reporter complex according to the invention possesses very high stability in its initial state under PCR conditions. The initial signal is comparable to that generated by a single-part DNA probe or a single-part 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-part detection molecules. This is the case even though this complex of a target sequence-independent modular reporter is dissociated with each PCR cycle and must be reformed before signal readout.

[0057] 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, it offers entirely new possibilities for detecting and differentiating multiple DNA sequences in the same channel of a PCR detector.

[0058] In embodiments of the method according to the invention, steps c. to h. are carried out in the context of a reaction selected from the group comprising PCR, digital PCR, RT-PCR, digital RT-PCR, real-time / qPCR, droplet PCR, or any combination thereof.

[0059] In one embodiment of the method according to the invention, steps c. to h. are carried out within the framework of a PCR reaction, preferably a digital PCR reaction.

[0060] In other embodiments of the method according to the invention, steps c. to h. are carried out within the framework of a PCR reaction, preferably a real-time PCR or qPCR reaction.

[0061] In some of the aforementioned embodiments of the method according to the invention, steps c. to h. are carried out within the framework of a droplet PCR or emulsion PCR reaction.

[0062] These diverse application possibilities 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. This represents a significant improvement over the state of the art.

[0063] 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 oligo, at least one base strand, at least one buffer, PCR polymerase.

[0064] 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. In embodiments, the kit according to the invention can be used to carry out the method according to the invention.

[0065] 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.

[0066] 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.

[0067] Detailed description of the invention

[0068] The term "target sequence-nonspecific reporter complex" describes a complex of target sequence-nonspecific nucleic acid oligonucleotides (e.g., DNA oligonucleotides) for signal generation during PCR in the presence of DNA target sequences. Preferably, a target sequence-nonspecific reporter complex comprises at least one label and at least two oligonucleotides, namely 1) a base strand comprising at least one mediator binding site and at least one signal oligo binding site, and 2) at least one signal oligo, wherein the signal oligo binding site of the base strand and the at least one signal oligo hybridize to each other but are not covalently linked.

[0069] 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 oligos and mediators, which thus form a signal 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, upon extension 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 base strand can comprise one or more signal complexes and one or more receptor complexes, whereby a signal oligo complex can comprise at least one signal oligo binding site and (in the non-activated state) at least one signal oligo, and a receptor complex can comprise at least one Mediator binding site.

[0070] 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.

[0071] In the context of the present invention, a "label" may describe one or more fluorophores or one or more quenchers. Accordingly, in the context of the invention, a base strand and / or a signal initiation molecule or signal oligo may, in embodiments, carry or comprise one or more fluorophores and / or quenchers. The proximity of a fluorophore to the quencher prevents the detection of its fluorescence, whereas degradation of the signal oligo by hydrolysis by the 5'-to-3' exonuclease activity of the PCR polymerase used for the amplification reaction disrupts the reporter-quencher proximity, thus allowing unquenched emission of fluorescence that can be detected upon laser excitation.In preferred embodiments, a target sequence-nonspecific modular reporter complex comprises at least one fluorophore and at least one quencher (these are therefore preferably present in pairs), wherein the quencher preferably suppresses the fluorophore signal as long as the signal oligo is hybridized to the signal oligo binding site, wherein the at least one fluorophore and the at least one quencher can be located to each other within the target sequence-nonspecific modular reporter complex either in the cis position (both either on the signal oligo or on the base strand) or in the trans position (one of the two labels is located on the base strand, the other on the signal oligo, or vice versa). In embodiments in which several quencher-fluorophore pairs are present within a target sequence-nonspecific modular reporter complex, the pairs can also be arranged in different locations relative to each other, e.g.some in cis, others in trans, or all in cis or trans. A base strand may comprise none, one, or more tags, for example, there may be none or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tags, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or even 25 tags. In some embodiments, a base strand comprises none, 1, 2, 3, or up to 5 tags. In other embodiments, a base strand comprises no, 1, 2, 3, 4, 5, or even more than 5 labels. A signal oligo may comprise no, one, or more labels, for example, no or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or even 20 labels, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 labels.In some embodiments, a signal oligo comprises no, 1, 2, 3, or up to 5 labels. In other embodiments, a signal oligo comprises no, 1, 2, 3, 4, 5, or even more than 5 labels.

[0072] Herein, a "mediator" refers to an oligonucleotide and part of the receptor complex, which can be extended along the "base strand" by the polymerase. A "mediator probe" describes a nucleic acid oligonucleotide, preferably a DNA oligonucleotide, which establishes the connection between the target sequence and the target-sequence-independent (modular) receptor by binding to it during PCR in the presence of a DNA target sequence, being cleaved by the exonuclease activity of the polymerase, and releasing a mediator sequence, which then binds to a base strand and, together with the base strand, forms a receptor complex.

[0073] For the purposes of the invention, a "C3 spacer" is preferably a chemical block group (protecting group), which preferably comprises a chain of three carbon atoms. This block group (protecting group) preferably serves to prevent (non-specific) polymerase extension of the strand. Typical and—depending on the embodiments—suitable C3 spacers / block groups (protecting groups) are known to the person skilled in the art, and the person skilled in the art knows, based on the present disclosure of the invention, how to select suitable C3 spacers / block groups (protecting groups) as routine adaptations of the invention described herein.

[0074] 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."

[0075] 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%.

[0076] 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 level of gene expression in a sample in real time. In qPCR, fluorescently 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. Real-time PCR makes it possible to monitor the progress of an ongoing amplification reaction as it occurs (i.e., in real time). Data is therefore collected throughout the entire PCR reaction and not at the endpoint as with conventional PCR.Measuring reaction kinetics in the early stages of PCR offers significant 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 in classical PCR. The higher the starting copy number of the nucleic acid target, the more likely a significant increase in fluorescence is to be 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. 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 then 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.

[0077] 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.

[0078] In the context of the invention, a “signal change” describes a fluorescence signal change.

[0079] This signal change is preferably a significant, differentiable, and / or characteristic change in the fluorescence signal, which is clearly distinct from or differentiated from potential baseline or background signals or background or background noise. Those skilled in the art are aware that under some experimental conditions, nonspecific fluorescence baseline signals or background or background noise can occur due to fluorophores during fluorescence detection. Therefore, a signal change in the context of the invention preferably describes a significant, differentiable, and / or characteristic change in the fluorescence signal, and not a fluorescence baseline or background signal or background or background noise. In preferred embodiments, this signal change can mean an increase in fluorescence intensity, in other words, an increase in the fluorescence signal. In some embodiments, a signal change is a decrease in the fluorescence signal.The increase in a fluorescence signal is preferably due to the fact that an amplification reaction increases the number of target sequence amplicons and thus the activation of associated signaling complexes. Accordingly, the number of resulting cleavages, digestions, and / or separations of the respective signal oligos from their binding site on the associated base strands increases, whereby at least one fluorophore is released and / or separated from its quencher (i.e., the distance between quencher and fluorophore increases such that the fluorescence signal is no longer quenched by the quencher). An increase (increase in the number) of released and / or non-quenched fluorophores thus leads to an increase in the fluorescence signal, which is specific and indicative for a specific target sequence.The more target sequences are present and bound by mediator probes in a PCR reaction, the more the fluorescence signal increases. Preferably, the fluorescence 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 or droplet PCR, preferably only one target sequence is present 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 max. 1 target sequence per reaction chamber (e.g.,Partition, emulsion droplets) so that when amplification begins and a similar amplification efficiency is achieved in all reaction compartments (containing a target sequence), the specific signal for the detection of an identical target sequence in different reaction compartments 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 compartment. In embodiments, the intensity / strength of a respective label, which is 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).

[0080] “Fluorophore” (or fluorochrome, similar to a chromophore) is a fluorescent chemical compound that can re-emit light when excited by light. Fluorophores for use as labels in the construction of labeled probes of the invention include, but are not limited to, rhodamine and derivatives such as Texas Red, fluorescein and derivatives such as 5-bromomethylfluorescein, Lucifer Yellow, IAEDANS, 7-Me2N-coumarin-4-acetate, 7-OH-4-CH3-coumarin-3-acetate, monobromobimane, pyrene trisulfonates such as Cascade Blue and monobromotrimethyl-ammoniobimane, 7-NH2-4CH3-25-coumarin-3-acetate (AMCA), FAM, TET, CAL Fluor Gold 540, JOE, VIC, Quasar 570, CAL Fluor Orange 560, Cy3, NED, Oyster 556, TMR, CAL Fluor Red 590, HEX, ROX, LC Red 610, CAL Fluor Red 610, Texas Red, LC Red 610, CAL Fluor Red 610, LC Red 640, CAL Fluor Red 635, Cy5, LC Red 670, Quasar 670, Oyster 645, LC Red 705, Cy5.5, BODIPY FL, Rhodamine Green, Oregon Green 30 488, Oregon Green 514, Cal Gold, BODIPY R6Gj, Yakima Yellow, Cal Orange, BODIPY TMR-X, JOE, HEX, Quasar-570 / Cy3, TAMRA, Rhodamine Red-X, Redmond Red, BODIPY 581 / 591 , Cy3.5, Cal Red / Texas Red, BODIPY TR-X , BODIPY 630 / 665-X, Quasar-670 / Cy5, Pulsar-650, Dy490, Atto-488, Atto532, Atto-Rho-6G, Dy590, Atto-Rho101 , Cy5, Dy-636, Atto-647N, Cy5.5, Dy682, Atto-680, BMN-488, BMN-505, BMN-536, BMN-562,.

[0081] "Quenching" refers to any process that reduces the fluorescence intensity of a given substance. Quenching is the basis for Förster resonance energy transfer (FRET) assays, static or contact quenching assays, or a combination of both. FRET is a dynamic quenching mechanism because the energy transfer occurs while the donor is in an excited state. Contact quenching requires close spatial proximity in the form of physical contact between the donor and quencher. A "quencher" is a molecule that quenches the fluorescence emitted by the fluorophore when it is excited by the light source of a PCR cycler or detection device.Quenchers for use as labels in the construction of labeled signal oligos and / or base strands of the invention include, without claiming to be exhaustive, DDQ-I, Iowa Black, Iowa Black FQ, QSY-9, BHQ-1, QSY-7, BHQ-2, DDQ-II, 22 Eclipse, Iowa Black RQ, QSY-21, BHQ-3 Dabcyl, QSY-35, BHQ-0, ElleQuencher, BMN-Q1, BMN-Q2, BMN-Q60, BMN-Q-535, BMN-Q590, BMN-Q620, BMN-Q650. The person skilled in the art knows suitable reporter-quencher pairs and knows which to select for a particular application.

[0082] Implementation examples

[0083] 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.

[0084] Description of the methods and implementation of the examples

[0085] For the examples corresponding to the results shown in Fig. 6, a real-time Mediator probe PCR was performed on a Rotor-Gene Q 60006-plex from Qiagen. The temperature profile consists of an initial denaturation for 2 minutes at 95 °C followed by 45 cycles (1 cycle corresponds to: 10 seconds at 95 °C, 80 seconds at 58 °C, and a further 10 seconds at 58 °C for fluorescence readout).

[0086] Another embodiment involves performing a digital PCR in the QIAcuity system from Qiagen according to the results shown in Fig. 7. The temperature profile results from an initial denaturation over a period of 2 min. This is followed by 40 repetitions of a cycle consisting of 15 seconds of denaturation at 95 °C followed by a subsequent annealing step at 58 °C for 30 seconds.

[0087] To generate the results as shown in Figure 9 of the multiplex variant example, a digital multiplex Mediator Probe PCR can be performed using three different Mediator probes for three different target sequences, whereby the following three complexes, each consisting of a base strand and one or two signal oligos, are used as modular reporter complexes for the three respective Mediator probes:

[0088] Modular reporter complex 1: Sequence ID 11 (SEQ ID NO: 11), Sequence ID 12 (SEQ ID NO: 12)

[0089] Modular reporter complex 2: Sequence ID 13 (SEQ ID NO: 13), Sequence ID 14 (SEQ ID NO: 14)

[0090] Modular reporter complex 3: Sequence ID 15 (SEQ ID NO: 15), Sequence ID 11, Sequence ID 13 In embodiments, the digital PCR can be carried out analogously to the experimental description of the embodiment for digital PCR and to the procedure according to Experiment 7, whereby a fluorescence signal must be measured in at least two suitable wavelength ranges in order to be able to obtain and evaluate the signal patterns shown in Table 2.

[0091] Procedure in the experiment for Fig. 6: For a reaction volume of 25 μl per tube, the fluorescence was measured in the green channel (wavelength: excitation at 470 nm, detection at 510 nm) with optimized gain settings for NTC (without target) and PTC (with target) duplicates. Plasmid DNA from Haemophilus ducreyi (Hd for short) was chosen as the target, of which 2.5 pg were added per single reaction (PTC). The primers used were obtained from Biomers.net GmbH, Ulm, Germany, and are listed in Table 1. The PerfeCTa Multiplex qPCR ToughMix from Quantabio, Beverly, Massachusetts, was used in 1-fold. The forward and reverse primers for Hd [Sequence ID: 08 and 09] were used in 0.1 pM as well as 0.2 pM of the corresponding mediator probe [Sequence ID: 10]. Within the experiment, the state-of-the-art Universal Reporter (UR) was compared with the bipartite reporter and two multiplex variants were tested.The concentrations of the varying fluorescence-generating probes for the individual reactions were selected as follows: The UR (Sequence ID (SEQ ID NO): 01) was used at 0.05 pM, and the split reporters consisting of signal oligo and base strand [Sequence ID (SEQ ID NO): 02 and 03] were each used at 0.08 pM. For the two multiplex variants, 0.05 pM each of the two signal oligos [Sequence ID (SEQ ID NO): 05 and 06] and 0.1 pM of the base strand oligo [Sequence ID (SEQ ID NO): 04 and 07] were used. Those skilled in the art will understand how to conduct the experiment based on the specified parameters, primer sequences, and concentrations. The resulting curve is shown in Fig. 6.

[0092] Procedure in the experiment for Fig. 7: For the digital PCR, the QIAcuity Probe Mix from Qiagen was used at a 1x concentration with internal reference dye. The target and its concentration are identical to the previously described example. The sequences can be found in Table 1. 0.8 pM of each of the forward and reverse primers [Sequence ID: 08 and 09] were used, as well as the mediator [Sequence ID (SEQ ID NO: 10] at a concentration of 1.6 pM. A concentration of 0.4 pM per reaction was chosen for both the universal [Sequence ID (SEQ ID NO: 01]] and the split reporters [Sequence ID (SEQ ID NO: 02 and 03]. A 26K 24-well nanoplate was filled with 40 pL reaction volume per well. Fluorescence was measured on the QIAcuity system in the green color spectrum (excitation: 463-503 nm, emission: 518-548 nm) using the standard settings known to those skilled in the art. The results for the experiment are shown in Fig.The results shown in Figure 7 were evaluated using absolute quantification with the QIAcuity Software Suite 1.2.18. A1 corresponds to the NTC, A2 to the PTC for the state-of-the-art (UR Green, [Sequence ID (SEQ ID NO): 01]). Wells C1 and C2 show the results of the NTC and PTC for the split reporter system [Sequence ID (SEQ ID NO): 02 and 03], respectively. Table 1: Primer sequences used for the exemplary embodiments with designation and (internal) modification. The sequences for the multiplex variants are used only for the exemplary embodiment of the experiment in Figure 6.

[0093]

[0094] Table 2: Primer sequences used with designation and (internal) modification for the embodiment (multiplex detection) according to the method shown in Fig. 9.

[0095]

[0096] Two-part reporter for optimizing fluorescence signals

[0097] The target sequence-nonspecific modular reporter complex can, in some embodiments, consist of a base strand with a label at the 5' end and a signal initiation strand with a label at the 3' end (Figure 4B). A fluorophore and a quencher are attached to each labeling site, which means that no fluorescent signal is generated in the initial state. By extending the mediator during the PCR reaction by the PCR polymerase, preferably with exonuclease activity, the bond between the base strand and the signal strand (signal oligo) is broken, or the fluorophore's bond is broken, thus generating a signal.

[0098] Both the quenching efficiency and the fluorescence intensity of various dyes can be easily optimized for PCR applications, as only one strand needs to be exchanged at a time and can also be used directly (Figure 4B). The system is ideally suited for this purpose, as the behavior and performance parameters of the modifications and labels can vary slightly depending on the sequence and attachment variant. Furthermore, the target sequence-unspecific modular reporter complex used in this way is significantly cheaper than state-of-the-art probe systems and performs at least as well as state-of-the-art single-part universal reporters in PCR and digital PCR. This was demonstrated by the results of comparative experiments, as shown in Figures 6 (PCR analysis) and 7 (dPCR analysis).The target sequence-independent modular reporter complex according to the invention therefore allows the combination of fluorophores and quenchers to be optimized much more efficiently compared to a single-part doubly labeled oligonucleotide (state of the art).

[0099] In the present experiment, the split target sequence-unspecific reporter for optimizing fluorescence signals demonstrates very good and improved performance, both compared to the currently used state-of-the-art universal reporter and with regard to its use in investigating optimal fluorophore-quencher combinations. The results of a first comparative experiment, presented in Figure 6, show that the split system surprisingly functions excellently despite the non-covalent bond between fluorophore and quencher across multiple nucleotides of the same molecule. As can be seen in Figure 6, the curve of the split modular reporter with a quencher-labeled base strand and a fluorophore-labeled signal initiation strand (Figure 6, curve with crosses) and the curve of the state-of-the-art universal reporter (Figure 6, curve with triangles) exactly overlap.The system according to the invention not only enables the successful detection of target sequences, but also enables the simultaneous detection of multiple target sequences. The result thus demonstrates that multiple fluorophores and quenchers can be used to vary the base signal and thus, for example, to multiplex monochrome (e.g., using the same fluorescence color). This is illustrated by the curve of the reporter complex according to the invention with one quencher and two fluorophores (Figure 6, curve with circles, "Multiplex variant 1") and the curve of the reporter complex according to the invention with two quenchers and two fluorophores (Figure 6, curve with triangles, "Multiplex variant 2"), each in duplicate.

[0100] Another surprising result is the optimal functioning of the bipartite reporter in digital PCR (dPCR). An example of this is shown in the experimental result in Figure ? and includes a digital PCR test in the green detection channel by comparing the bipartite reporter (Figure ?, bars "C17"C2") to a state-of-the-art Universal Reporter (UR) (Figure ?, bars "A17"A2"). Both the negative amplification control and the positive amplification control show that the inventive system successfully detected the same amount of positive droplets as the state-of-the-art Universal Reporter (UR).

[0101] Base strand with double-labeled signal initiation strands

[0102] The use of an unlabeled base strand also offers advantages. Firstly, it simplifies and reduces the cost of production. Secondly, various signal initiation molecules (or "signal molecules" for short), which in this case carry a fluorophore and quencher, can be attached. For example, probe systems such as the TaqMan probe or molecular beacons can be attached to a base strand, whereby signal generation remains target sequence-nonspecific, but incorporates readily available state-of-the-art systems. The results of an example experiment using one embodiment are shown in Figure 8; the simplified functional diagram of this representative embodiment can be found in Figure 4A. By binding several doubly labeled signal oligos, for example, much stronger signal changes can be generated per complex than previously achieved with single-part DNA probes or state-of-the-art reporter molecules.Another surprising result was revealed by the use of an unlabeled base strand and a signaling oligo similar or equivalent to the TaqMan probe (Figure 8, curve with circles). It was demonstrated that the modular system according to the invention generates a very good signal in this case and can be successfully used to detect target sequences. This was also demonstrated in comparison to the state-of-the-art Universal Reporter (UR) in triplicates and is shown in Figure 8 (curve with triangles).

[0103] For direct multiplexing, i.e., the simultaneous detection of different target sequences in a sample and in a reaction, either multiple optical channels, additional processing steps, or complex concentration adjustment or modification of the reporter molecules are required, according to the current state of the art. In contrast, the reporter according to the invention offers the possibility of combined measurements across different channels, for which purpose basic strands according to the invention with more than one receptor complex can be used. In these embodiments, the receptor complexes are preferably arranged offset along the base strand so that they activate different signaling complexes (Figure 3). The possible different color and / or intensity combinations of the respective markers can preferably encode different extended mediator sequences and thus different detected target sequences.

[0104] In embodiments, multiple target sequences can be detected simultaneously using different fluorescence intensities by using different target sequence-nonspecific modular reporter complexes. These target sequence-nonspecific modular reporter complexes each have different mediator binding sites on the base strand, each with a different number of fluorescent and / or quencher labels. Various embodiments of this are shown in Figures 5A, B, and C. In this way, signals of different signal intensities are generated from each signal complex type, which can thus be distinguished in a fluorescence channel (see the results of an exemplary experiment in Figure 6). These embodiments offer significant advantages, particularly in digital PCR, for distinguishing different signal clusters and represent an improvement over the state of the art.

[0105] Figure 9 describes a further embodiment of such a multiplex variant, in which, in addition to two reporter complexes (Fig. 9A and B), each with a different mediator binding sequence and a different signal oligo binding site for a different signal oligo, each with a different fluorophore that generates a signal change in a different channel upon activation by the respective mediator, a third reporter complex (Fig. 9C) is added. This is activated by another different mediator, but also has two binding sites for the respective signal oligos of reporter complexes 1 and 2. In the prior art, corresponding reporter molecules, which have two fluorophore and two quencher molecule modifications as well as a 3'-block group, are very complex to produce and must also be newly synthesized for each new target sequence.In contrast, the synthesis of a modular reporter according to the invention is less complex and also offers the possibility of freely combining various signal oligos available in the laboratory with a base strand in the experimental setup without the need for further synthesis. This represents a clear advantage, e.g., with regard to optimization effort and costs, compared to the probe systems of the state of the art. Upon activation of this reporter complex, a simultaneous signal can be generated in both detection channels. In the case of digital PCR, where statistically only a single DNA copy is present in a reaction chamber (e.g.,Thus, with only two available color channels in a detection device, three DNA target sequences can be distinguished by using a mediator probe multiplex PCR according to the invention in the presence of the respective DNA sequence to cleave the respective mediator probe, thus releasing the corresponding mediator. It is clear to those skilled in the art how to carry out a corresponding digital PCR. Accordingly, the respective DNA sequence can be deduced from the respective change in the fluorescence signals: Table 3: Description of the multiplex approach, which results from the use of three modular reporter complexes that can bind two different signal oligos in different proportions.

[0106] Compared to the state of the art, the described method makes it much easier to generate corresponding signal patterns by using different combinations of a basic set of base strands and signal oligos.

[0107] Figures

[0108] The invention is further described by the following figures. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention provided to illustrate the invention described herein.

[0109] Figure 1: The figure shows the mediator probe cleavage during a PCR with activation of the mediator in one embodiment of the invention.

[0110] Figure 2: A) shows the signal generation by activation of the signal oligo on a target sequence-nonspecific modular universal reporter complex in one embodiment of the invention. B) shows the schematic structure of one embodiment of a target sequence-nonspecific modular universal reporter complex according to the invention.

[0111] Figure 3: The figure shows a target sequence-unspecific modular reporter complex with alternating signal and receptor complexes, which enable, for example, mediator signal coding for monochrome multiplexing.

[0112] Figure 4: The figure shows examples of different labeling positions of the fluorophore and quencher. A) shows an example of cis-labeling on the same signal oligo. B) shows an example of trans-labeling.

[0113] Figure 5: The figure shows exemplary embodiments for modeling the signal strength (fluorescence signal strength) by multiple labeling of a target sequence-nonspecific modular reporter complex or by using different quencher strengths. A) shows an example of cis-labeling, where a quencher and a fluorophore are present on each base strand and the signal oligo. B) shows another example of cis-labeling, where a quencher is present on the base strand and two signal oligos, each with a fluorophore. C) shows an example of cis-labeling, where two quenchers and two fluorophores are present on each base strand and signal oligo.

[0114] Comparison of the modular reporter according to the invention to a universal reporter

[0115] (UR) of the prior art (curve with triangles). The figure demonstrates the optimal functionality of the two-part reporter according to the invention (curve with crosses) compared to the UR. The advantageous properties of the reporter according to the invention also offer possibilities for multiplexing detection reactions, e.g., by using reporters according to the invention with labels of different fluorescence intensities, e.g., by one quencher and two fluorophores (multiplex variant 1, curve with circles) or by two quenchers and two fluorophores (multiplex variant 2, curve with squares). NTCs (non-template controls) are shown in dark gray.

[0116] Illustration of the experimental dPCR results of an embodiment of the bipartite reporter according to the invention compared to a prior art UR. The bipartite reporter according to the invention (NTC: C1, PTC: C2) showed quantitatively the same results as the UR (NTC: A1, PTC: A2) with regard to positive and negative dPCR droplets.

[0117] Figure 8 Comparison of the state-of-the-art universal reporter (UR) (curve with triangles) with the modular reporter according to the invention. Shown is an embodiment with an unlabeled base strand and a double-labeled signal oligo (curve with circles), which resembles a "TaqMan probe" (NTCs are shown in dark gray).

[0118] The figure shows a further embodiment of a multiplex variant according to the invention, in which in addition to two reporter complexes (A and B), each of which has a different

[0119] A mediator binding sequence and a different signal oligo binding site for each different signal oligo, each with a different fluorophore, which generates a signal change in a different channel upon activation by the respective mediator, are added. A third reporter complex (C) is also added. This is activated by another different mediator, but also has two binding sites for the respective signal oligos of reporter complexes 1 and 2.

[0120] References

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[0122] Faltin, Bernd; Zengerle, Roland; Stetten, Felix von (2013): Current Methods for Fluorescence- Based Universal Sequence-Dependent Detection of Nucleic Acids in Homogenous Assays and Clinical Applications. In: Clinical Chemistry 59 (11), S. 1567-1582. DOI:

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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 oligo binding site; ii. at least one signal oligo, wherein the at least one signal oligo binding site of the base strand and the at least one signal oligo hybridize 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-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 the at least one target nucleic acid sequence; e.a. Cleavage of the probe sequence of at least one mediator probe bound to at least one target nucleic acid sequence by a PCR polymerase with nuclease activity during PCR amplification, releasing the mediator sequence; b. Binding of at least one released mediator sequence to a mediator binding site of the at least one target sequence-nonspecific modular reporter complex; c. Extension of the sequence of at least one mediator sequence bound to a mediator binding site by a PCR polymerase, breaking the bond between the hybridized at least one signal oligo binding site and the at least one signal oligo, thereby initiating a signal change; d. Detection of at least one signal change as evidence of the at least one target nucleic acid sequence. A method according to claim 1, wherein the at least one label comprises at least one fluorophore and / or at least one quencher. A method according to claim 2, wherein no fluorescence signal change is generated by the at least one fluorophore when the at least one signal oligo is hybridized with the at least one signal oligo binding site of the base strand, wherein either the at least one quencher is localized at the at least one signal oligo binding site of the base strand and the at least one fluorophore is localized at the at least one signal oligo or vice versa, and wherein in step g. at least one fluorophore and at least one quencher are separated, thereby initiating a signal change.A method according to claim 2, wherein the at least one label comprises at least one fluorophore and at least one quencher, and wherein both the at least one quencher and the at least one fluorophore are localized on the at least one signal oligo, and wherein in step g. the at least one signal oligo is cleaved by PCR polymerase, thereby separating the at least one fluorophore and the at least one quencher, thereby initiating a signal change. A method according to any of the preceding claims, wherein the base strand comprises at least one signal oligo binding site to which two or more signal oligos are hybridized, and wherein the two or more signal oligos and / or the base strand have one or more labels at the at least one signal oligo binding site.A method according to any one of the preceding claims, wherein the base strand comprises two or more signal oligo binding sites, and wherein at least one of the signal oligos is hybridized to the two or more signal oligo binding sites and / or the base strand has one or more markers at at least one of the two or more signal oligo binding sites. A method according to any one of the preceding claims, wherein the base strand comprises two or more signal oligo binding sites which together form a signal complex and to which at least one signal oligo with at least one marker is hybridized, wherein a signal change generated by the markers of the signal complex is characteristic of a target sequence.Method according to one of the preceding claims, wherein the base strand comprises at least two or more signal complexes with different signal oligos and / or different markings on the signal oligos, and wherein the base strand comprises a mediator binding site corresponding to the at least two or more signal complexes.

9. A method according to any of the preceding claims, wherein the at least one target sequence-unspecific modular reporter complex enables the detection of at least one first and one second target nucleic acid sequence, comprising the basic strand, at least one first and one second mediator binding site for at least one first and one second mediator sequence of at least one first and one second mediator probe, and at least one first and one second signal oligo binding site to which at least one first and one second signal oligo is hybridized, and wherein the first mediator probe has a probe sequence with an affinity for a first target nucleic acid sequence and the second mediator probe has a probe sequence with an affinity for a second target nucleic acid sequence.

10. Method according to the preceding claim, wherein the base strand has at least one first and one second label, wherein the signal change due to the at least one first label is characteristic of the first target nucleic acid sequence, and the signal change due to the at least one second label is characteristic of the second target nucleic acid sequence.

11. Method according to one of the preceding claims, wherein in step a. at least one first and one second target sequence-nonspecific modular reporter complex is provided, wherein the at least first target sequence-nonspecific modular reporter complex enables the detection of at least one first target nucleic acid sequence and the at least second target sequence-nonspecific modular reporter complex enables the detection of a second target nucleic acid sequence, wherein the signal change due to the at least one label of the at least first target sequence-nonspecific modular reporter complex is characteristic of the first target nucleic acid sequence and the signal change due to the at least one label of the at least second target sequence-nonspecific modular reporter complex is characteristic of the second target nucleic acid sequence.

12. Method according to the preceding claim, wherein the signal changes characteristic of the at least first and the at least second target nucleic acid sequence differ from each other by their color and / or their fluorescence or signal strength.

13. A method according to any of the preceding claims, wherein steps c. to h. are carried out in a reaction selected from the group comprising PCR, digital PCR, RT-PCR, digital RT-PCR, real-time / qPCR, droplet PCR, or any combination thereof.

14. Method according to 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.

15. 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 oligo, at least one basic strand, at least one buffer PCR polymerase.

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