PCR multiplexing with target sequence-independent reporter molecules with distinguishable signal intensities.

Abstract

The present invention relates to a method for specifically detecting at least two nucleic acid target sequences in the same detection channel by at least two target sequence-independent reporter molecules, in which at least two mediator probes comprise at least one probe sequence and one mediator sequence, at least two target sequence-independent reporter molecules of a first type and a second type are used, each of which comprises at least one label having a respective maximum signal intensity in the same detection channel, and at least two nucleic acid target sequences are detected by a nucleic acid detection reaction due to their characteristic signal intensity and / or emission spectrum. The present invention also relates to a kit for carrying out the method.

Description

[Technical field]

[0001] In recent years, as a further development of real-time PCR, especially digital PCR, molecular diagnostics has been revolutionized by rigorous and sensitive nucleic acid detection methods, because these methods are superior to standard PCR in detecting rare genetic targets (Non-Patent Document 1, Non-Patent Document 2). In digital PCR (dPCR), the reaction mix and the nucleic acids contained therein (e.g. ctDNA or DNA) are divided into thousands of reaction spaces, which separates the target sequences from each other and facilitates their amplification and detection, especially for rare target sequences. A common method here is, for example, to microfluidic dripping the reaction mix with the nucleic acid to be detected into oil or dispensing it into fixed microcavities during PCR, thereby forming a closed reaction space for amplification and detection with each formed unit. If the nucleic acid target sequence is present in the droplet, it is amplified by PCR (or other amplification method). The individual amplicons are usually detected by sequence-specific fluorogenic nucleic acid probes, which generate a corresponding fluorescent signal. In addition to absolute quantification, dPCR also has the advantage of reducing the influence of inhibitors. Since quantification in digital PCR is performed as an end-point analysis, "signal clusters" (also called populations) in the data space can be assigned to specific DNA or cDNA target sequences by the strength of the fluorescent signal, thereby enabling alternative types of data classification (Non-Patent Document 3, Non-Patent Document 4).

[0002] Multiplexing is one of the key aspects of dPCR, since various nucleic acid target sequences are often present in a sample at very low concentrations, making it crucial to detect as many target sequences as possible in the sample. The most common multiplexing method for dPCR is based on the fact that DNA reporter molecules generate a fluorescent signal for each DNA target sequence within a spectral range corresponding to the detection range of the detection channel. Thus, one DNA target sequence can be detected per detection channel on the device. However, the degree of multiplexing of the device is initially limited by the number of detection channels available. Currently available dPCR devices on the market have two to six different fluorescent detection channels, which means that up to six target sequences can be analyzed on the device. The main problem here is that wavelengths may overlap with many detection channels, which may result in low specificity and sensitivity due to the resulting crosstalk.

[0003] One way to increase the degree of multiplexing is to use a combinatorial approach, for example by combining Taqman probes and DropOff probes as target sequences of specific reporter molecules. In this case, the combinatorial logic of the signals generated by these probes leads to a similarly high degree of multiplexing (Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7). These methods are also very complex to develop, require a great deal of effort to evaluate, and are correspondingly complex to qualify or move to new target panels, since individual mutations may only be detected indirectly.

[0004] Furthermore, there are other methods to increase the degree of multiplexing of digital PCR, aiming to generate additional populations in the data space. US Patent No. 5,339,363 by Bio-Rad describes multiplex digital PCR analysis of more target sequences than available optical channels, taking into account other sample-specific aspects. However, the patent does not disclose any new assay features or methods to generate such target sequence populations. According to current knowledge, these methods can essentially only be used when the droplets are clearly multiply occupied by different DNA target sequences, or the signal is generated by target sequence-specific reporter molecules such as Taqman probes or non-sequence-specific intercalating dyes, but they do not represent an innovation over the prior art. It is described that different target sequence-specific probes can, for example, use different fluorophores as labels for this purpose, or can have multiple fluorophore labels so that multiple populations are generated in the same detection channel. Since the signals of the probes described herein are suppressed to the ground state with low efficiency by FRET quenching, the initial signal suppression is relatively low, which is why effective modeling of the signal strength and therefore separation of the populations in the data space is only possible to a limited extent. These labeled probes are also target sequence specific, so that extensive optimization of the fluorescent properties of these labeled probes is very inefficient, since a new labeled probe needs to be synthesized every time and the procedure needs to be restarted when moving to another sequence. Similarly, multiple modification of such target sequence specific probes with fluorophores is very complicated, and they also need to be modified with additional quenchers to suppress the signal sufficiently to the ground state to avoid non-specific signal populations entering the data space. Such multiple modifications are rarely used as target sequence specific reporter molecules, since the synthesis is very complicated and usually requires extensive optimization, and the procedure is very inefficient.

[0005] Therefore, the most common variant of this intensity multiplexing is to vary the concentration of different types of target sequence-specific reporter molecules (e.g., Taqman probes) while maintaining a uniform and simple fluorescent label per detection channel (Figure 1 right side) (Non-Patent Document 8, Non-Patent Document 4). This allows the creation of additional populations in the data space of the graph. Due to the use of target sequence-specific fluorogenic reporter molecules, the signal generation depends directly on the target sequence or the PCR system. This direct detection therefore poses the problem of a large optimization effort, since the large variance (the presence of data points between different populations) leads to unclear signal populations that reduce the precision (intensity multiplexing). The individual PCR components, especially the fluorogenic reporters with the same fluorescent label, must be well matched to each other, for example, in orthogonal concentration ratios, which is not always possible in practice. This method can also be supplemented by combining with other dyes that have an emission maximum between the different fluorescent detection channels and therefore exhibit additional fluorescent labels that can be equally detected in multiple fluorescent channels. This leads to corresponding clusters among other clusters in the data space, which becomes more and more difficult due to the expansion of the dimension of the data space due to the additional detection channels. This makes the development of corresponding multiplex detection, especially validation, very complicated. Another variation of intensity multiplexing is to use more complex concentration variations to correct the clusters in the data space, and therefore less susceptible to dispersion (Non-Patent Document 9).

[0006] In contrast, mediator probe PCR is a method for detecting nucleic acids that does not use target sequence-specific reporter molecules and is based on separating nucleic acid detection by a target sequence-specific mediator probe from signal generation by a target sequence non-specific universal reporter, and was patented by the University of Freiburg in 2012 (Patent Document 2, Non-Patent Document 10). This method is designed for colorimetric multiplexing with standardizable reporter molecules. A signal for each detection channel is recorded, which can be assigned to only one activated universal reporter in a homogeneous reaction, regardless of the signal strength. It has already been observed that under real-time PCR conditions, different fluorophores lead to different signal intensities and signal curves, and that by maximizing the signal generation of the target sequence non-specific fluorogenic universal reporter molecule, the performance characteristics of the corresponding multiplex real-time PCR can be optimized (Non-Patent Document 11, Non-Patent Document 12). However, since signal curves in the same detection channel cannot be distinguished using this approach, modeling the signal strength in real-time PCR has no practical added value. For colorimetric digital multiplex mediator probe PCR, it has also been shown that maximizing fluorescent signal generation results in better separation of populations in data space compared to negative controls (Non-Patent Document 13).

[0007] At the same time, a technology based on the separation of DNA sequence detection and signal generation by two types of detection molecules in real-time PCR was developed by Seegene Inc. In the above technology, an unlabeled PTO probe binds to a DNA target sequence, is cleaved, and fragments are released, which then form an extended duplex with a fluorescently labeled target sequence non-specific detection molecule (CTO molecule). This process is used to affect the signal generation across these detection molecules of different lengths. This allows, for example, to distinguish multiple target sequences in the same detection channel by reading the signal at a defined, predefined temperature. In another patent, reading at different temperatures in real-time PCR by one method in the same detection channel is also used, and therefore can be interpreted as a further development of the above-mentioned technology by Seegene (Patent Document 3). Here too, the extended reporter molecule is melted after detection. In contrast to the previous patent, the reporter molecule can be used to distinguish different target sequences. Currently, it is not possible to directly transfer this to dPCR, since temperature control during reading cannot be performed in dPCR in commercially available devices.

[0008] Increasing the degree of multiplexing in PCR by generating virtual fluorescence channels by photobleaching has already been addressed by Hahn-Schickard (Patent Document 4) (Non-Patent Document 14). To combine this approach with the advantage of separating signal generation and detection in digital mediator probe PCR, a step was added to the digital mediator probe PCR in which various universal reporter types with different fluorophore labels are used, bleached by light from an LED lamp (bleaching), and read in the same fluorescence detection channel. Depending on the fluorophore selected, a decrease in signal intensity can be observed as a result of bleaching, thereby separating populations in the data space of the chart and generating additional, so-called virtual channels (Non-Patent Document 15). However, the bleaching technique requires additional devices and process steps that cannot currently be provided by commercially available devices, making it impossible to implement in existing dPCR platforms due to technical and regulatory obstacles. However, in this approach, the fluorophores selected as labels for the universal reporters did not yet result in different fluorescent signal intensities before the bleaching step, which allowed uniform separation of populations in the data space of the diagram when more than one target sequence was included per reaction. The universal reporters used did not have the necessary configuration of fluorophores and quenchers that allowed single-color identification. Due to the high scattering of the fluorescent signal intensity of the reporter labels, a clear distinction could not be made between a single fluorescent signal population and a second signal population with a larger "dispersion" in the data space. In particular, this technique does not allow an unambiguous assessment of whether the population of a multiplex reaction is one or two, and therefore does not offer the possibility of direct multiplexing with target sequence non-specific reporters in the same detection channel (Non-Patent Document 15). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 9921154

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[0011] The prior art still lacks a method to adjust the signal intensity of target sequence-independent reporter molecules (e.g., generated by different fluorophores and / or quenchers) in one reaction to generate multiple distinguishable signal clusters in the same detection channel or corresponding regions of multidimensional data space, thereby taking advantage of the advantages of indirect signal generation in terms of assay accuracy and robustness to avoid previously described problems with colorimetric multiplexing or target sequence-specific intensity multiplexing in digital PCR. All that has been described so far is to monochromatically multiplex non-specific reporter molecules with target sequences in digital amplification, thereby achieving a higher degree of multiplexing by introducing additional process steps (e.g., bleaching). This complicates the process or evaluation, reduces accuracy, causes additional optimization efforts, and complicates, for example, approval procedures. Therefore, the object of the present invention is to increase the number of detectable nucleic acid target sequences in a detection reaction beyond the number of device-side detection channels. [Means for solving the problem]

[0012] The invention achieves this object by means of the methods of the dependent and independent claims.

[0013] Thus, in one embodiment, the present invention provides a method for specifically detecting at least two nucleic acid target sequences in the same detection channel by at least two target sequence-independent reporter molecules, comprising: a. providing at least a first nucleic acid target sequence and a second nucleic acid target sequence; b. providing at least a first mediator probe and a second mediator probe, each comprising an oligonucleotide; a first mediator probe oligonucleotide comprising a mediator sequence and a probe sequence, the mediator sequence having affinity for a first type of target sequence-independent reporter molecule, and the probe sequence exhibiting affinity for a first nucleic acid target sequence; the second mediator probe oligonucleotide comprises a mediator sequence and a probe sequence, the mediator sequence having affinity for a second type of target sequence-independent reporter molecule, and the probe sequence exhibits affinity for a second nucleic acid target sequence; at least the first mediator probe and the second mediator probe are label-free or preferably signal-generating label-free; c. Providing at least two target sequence-independent reporter molecules of at least a first type and a second type, each of which comprises at least one label having a measurable signal in the same detection channel and a nucleic acid sequence having a specific affinity to at least one mediator sequence, each of the at least two target sequence independent reporter molecule types is preferably unique to each type of target sequence independent reporter molecule by its at least one label, characterized so as to have a signal intensity distinguishable from the signal intensities of the labels of all other target sequence independent reporter molecule types and to be directly assignable to a respective nucleic acid target sequence; d. performing a nucleic acid detection reaction, wherein upon binding of the probe sequence of the at least first mediator probe to the at least first nucleic acid target sequence, at least one mediator sequence of the at least first mediator probe is released; At least one released mediator sequence binds to at least one first type of target sequence-independent reporter molecule, and a signal is generated that is characterized by a signal intensity and / or emission spectrum for a first nucleic acid target sequence bound by at least one probe sequence of at least one first mediator probe, the signal intensity and / or emission spectrum being assigned to each target sequence-independent reporter molecule by at least one label; Optionally, at least one mediator sequence of the at least one second mediator probe is released upon binding of the probe sequence to at least one second nucleic acid target sequence, and the at least one released mediator sequence binds to at least one second type of target sequence-independent reporter molecule, and a signal is generated from the at least one label, the signal intensity and / or emission spectrum of which is assigned to a respective target sequence-independent reporter and is characteristic of the second nucleic acid target sequence to which the at least one probe sequence of the at least one second mediator probe is bound; e. detecting the signal(s) generated under step d., comprising detecting the signal(s) in a detection channel and / or analysing the signal(s), the signal intensity(s) and / or the signal emission spectrum(s) and / or the cluster(s) of generated signals in the data space accordingly, preferably also being able to distinguish signals of different target sequence non-specific reporter molecules by their signal intensity in the same detection channel or corresponding regions of the data space; The present invention relates to a method comprising the steps of:

[0014] In an embodiment, therefore, the first mediator probe comprises an oligonucleotide that itself comprises a mediator sequence and a probe sequence. Thus, in an embodiment, the second mediator probe further comprises an oligonucleotide that itself comprises a mediator sequence and a probe sequence. It is preferred that the mediator sequence of the first mediator probe is different from the mediator sequence of the second mediator probe. It is preferred that the probe sequence of the first mediator probe is different from the probe sequence of the second mediator probe.

[0015] In other words, in an embodiment of the method according to the present invention, the mediator sequence of the first mediator probe is different from the mediator sequence of the second mediator probe, and the probe sequence of the first mediator probe is different from the probe sequence of the second mediator probe, respectively. Therefore, it is preferred that the probe sequence of the second mediator probe also has affinity for a "second" nucleic acid target sequence that is different (e.g., has a different nucleic acid sequence) from the "first" nucleic acid target sequence for which the probe sequence of the first mediator probe has affinity. The same applies to further (third, fourth, etc.) mediator probes that are optionally present and optionally used.

[0016] In an embodiment, step c. comprises providing at least two target sequence-independent reporter molecules of at least a first type and a second type, each comprising a nucleic acid sequence having a specific affinity for at least one label and at least one mediator sequence, each having a respective measurable signal in the same detection channel and / or a respective maximum signal intensity in the same detection channel.

[0017] According to the present invention, the "first" and "second" nucleic acid target sequences preferably differ in their nucleic acid sequence and / or their epigenetic modifications. The same applies to further (third, fourth, etc.) nucleic acid target sequences, optionally present and optionally detectable.

[0018] In a preferred embodiment of the method according to the invention, target sequence-independent reporters in the form of labeled oligonucleotides are assigned to nucleic acid target sequences. These are activated by the release of further oligonucleotides as part of the nucleic acid detection reaction, thereby generating a signal. The different types of reporter molecules are each modified with a different label (e.g. fluorophore and / or quencher and / or a different number of fluorophores and quenchers) and have different DNA sequences, which indirectly allows assignment to the target sequence. In the same detection channel, different target sequence-independent reporter molecule types generate signals of different signal strengths due to their different labels. As a result, the signals of different reporter molecule types, each with different labels, can also be distinguished based on different signal intensities in the same detection channel or in a corresponding spanning data space (axes in a multidimensional representation or a data space spanned by different detection channels in a purely mathematical comparison / analysis and / or algorithmic analysis) and can be assigned to the target sequence. According to the invention, it may be preferred to detect the signals of at least two different reporter complexes in the same detection channel or in corresponding regions of the data space. The fact that these reporter molecules are target sequence independent makes it possible for the first time to develop reporter molecules with particularly well distinguishable signal strength in the same detection channel. Because there is target sequence independence, these reporter molecules can also have suitable label configurations that allow efficient signal modeling. For example, the signal of a reporter molecule can be suppressed by contact quenching in the initial state, whereby the reporter molecule shows a large difference in signal generation in the activated state, or it can have multiple labels with fluorophores and / or quenchers. This allows users to increase the number of target sequences detectable in a reaction beyond the number of detection channels without the need to purchase additional equipment, expand the equipment, or perform further complicated process steps, which is a great added value and ensures high cost efficiency.Existing laboratory processes can be preserved and already-qualified devices can continue to be used without the need for reimplementation and, if necessary, requalification. Furthermore, this allows for efficient optimization of these target sequence-independent reporter molecules, which can then be used for further target sequences, without the need to develop and optimize them from scratch depending on the target sequence. Thus, the present invention provides a simplified and efficient process optimization that allows standard detection of different target sequences, reducing the effort and cost required.

[0019] The method according to the invention allows for rigorous and efficient (direct) single-color multiplexing with target sequence non-specific reporter molecules. The method according to the invention thereby allows for efficient target sequence independent optimization of reporter molecules, thus improving the separation of signal populations in the same detection channel or corresponding area of ​​data space. This allows for rigorous detection of additional nucleic acid target sequences beyond the number of detection channels of the device. This is because the use of target sequence independent reporter molecules greatly simplifies and improves the modeling of symmetric multiple labeling with fluorophores and quenchers, and / or labeling with different fluorophore labels in the appropriate configuration for the same detection channel, and / or fluorescent signal generation, for example by contact quenching, compared to target sequence specific reporter molecules.

[0020] This is done using target sequence-independent reporter molecules (e.g. universal reporters), which are labeled oligonucleotides that can each be (indirectly) assigned to a nucleic acid target sequence one wishes to detect and that are activated by binding of further oligonucleotides released as part of the nucleic acid detection reaction (e.g. PCR or digital PCR). In order to discriminate between signals that represent / indicate the presence of different nucleic acid target sequences in the same detection channel or corresponding regions of the data space (graphical representation of the detected signals), at least two target sequences one wishes to detect are each assigned to two different types of target sequence-nonspecific reporter molecules that generate signals of different signal strengths in the same detection channel.

[0021] The advantage of the method according to the present invention is the possibility of simultaneously detecting multiple target nucleic acid sequences in the same detection channel of a (PCR) device, for example, the channel that detects red fluorescent signals. This simultaneous detection in a single detection channel is realized by using different target sequence non-specific reporter molecules, each of which contains a label that has a similar fluorescence emission spectrum and generates a different signal that can be detected simultaneously in the same fluorescent detection channel of a device, for example, a digital PCR device, and is still distinguishable from each other. This feature allows the present invention to perform single-color multiplexing, i.e., the detection of multiple targets in a single ("monochromatic") fluorescent detection channel (with a specific wavelength range).

[0022] In some prior art digital PCR methods, the first or second target sequence is detected in emulsion droplets, each isolated in one droplet, by using two labeled probes, each generating a uniform signal that can be distinguished from non-specific amplification. In the method according to the invention, no labeled probe is used, which has the advantage that the reporter molecule according to the invention can be subsequently used in further assays, and thus the labeled reporter molecule can be optimized to a greater extent than is possible with known methods of the prior art. Furthermore, it may be preferred that the reporter molecule according to the invention only takes one configuration in which contact quenching occurs. This preferred property preferably enables or supports (direct) single-color multiplexing in the form according to the invention.

[0023] A further advantage of the method according to the invention is that the desired detection properties can be precisely adjusted by selecting each reporter molecule used, whose detection is independent of the target sequence. The connecting part between reporter molecules with the desired properties (e.g., fluorescence intensity and / or color) is a mediator probe, which is easy to make and design, and comprises a probe sequence and a mediator sequence. All that is required to prepare is two different sequence components of the mediator probe to individualize the target nucleic acid to be detected. These can then be combined with a target sequence non-specific reporter molecule that matches the mediator sequence. This independence allows the desired specific contact quenching to be set, which allows multiple distinguishable reporter molecule signals to be detected in the same detection channel. Moreover, signal optimization can be performed once, independent of multiple target sequences. Moreover, the method according to the invention is resource-conserving, since the reporter molecule set can be reused for any number of nucleic acid target sequences, making the initial one-off development of combinable reporter molecule signals very labor- and cost-efficient.

[0024] Furthermore, in the method according to the invention, the nucleic acid target sequences can be directly assigned to the data population (signal population in the evaluation / analysis) and no combinatorial theory is required, thus allowing the direct detection of the nucleic acid target sequences.

[0025] One possibility for generating different signal strengths by differential labeling of reporter molecules results from the physical molecular properties of the labels, which are amplified, for example, by contact quenching after activation of target sequence-nonspecific reporter molecules.

[0026] Therefore, in an embodiment, at least one label of the target sequence-independent reporter molecule is at least one fluorophore and / or at least one quencher.

[0027] Another essential characteristic of these target sequence non-specific reporter molecules in the efficient generation of strictly distinguishable signal populations is to select a labeling configuration that allows the signal intensity to be sufficiently robust and stable, and therefore to be generated sufficiently strictly so that it remains distinguishable without further process steps (such as bleaching) in the reading process.This has been shown to be possible, for example, by using a configuration corresponding to the basic structure of a universal reporter or a corresponding modification in a standardized manner.Appropriate modeling of target sequence non-specific reporter molecules can be performed, which allows the intensity of different signals in detection channels to be precisely adjusted (Non-Patent Document 11).

[0028] Consequently, in embodiments, different target sequence independent reporter molecule types differ in the signal intensity and / or emission spectrum of their at least one label.

[0029] In an embodiment, at least one label of the target sequence independent reporter molecule comprises at least two fluorophores and / or two quenchers with the same or different emission spectra and / or the same or different signal intensities.

[0030] In some embodiments, the first type of reporter molecule and at least the second type of reporter molecule differ in the number of labels or the number of fluorophores and / or quenchers. Thus, different types of target sequence-independent reporter molecules can be distinguished by the intensity of the signal and / or the intensity of fluorescence that they release when activated. For this purpose, different types of target sequence-independent reporter molecules can include different numbers and / or different fluorophores (with different emission spectra / colors and / or intensities) and / or quenchers or any combination thereof.

[0031] In a preferred embodiment in which the label comprises at least one fluorophore, the fluorescent signal is preferably suppressed by contact quenching until the reporter molecule is activated. Thus, preferably, no signal is released from a reporter molecule that is not activated. Activation preferably occurs by binding of the mediator sequence to a corresponding target sequence-independent reporter molecule and / or by subsequent polymerase-mediated extension of the mediator sequence already bound to the target sequence-independent reporter molecule during the nucleic acid detection reaction.

[0032] Thus, in embodiments, at least one label of a target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher, and preferably contact quenching occurs between the at least one fluorophore and the at least one quencher unless the mediator sequence is bound to the corresponding target sequence-independent reporter molecule or unless the bound mediator sequence is extended during the nucleic acid detection reaction.

[0033] This allows, for example, to detect additional DNA target sequences in a suitable device as part of digital DNA amplification and fluorescence-based detection without the need for further technical assistance or the need to vary the concentration of reporter molecules with fluorescent labels. This allows, for example, to obtain results similar to those produced by the combination of digital mediator probe PCR (MP-PCR) and photobleaching produced without the need for a bleaching process (e.g. by LED or heat), which significantly reduces and simplifies the experimental effort and allows implementation in existing devices. The method according to the invention therefore represents a very high added value compared to digital mediator probe PCR (MP-PCR) or other prior art methods, since it is directly compatible with existing platforms and can significantly simplify the approval of a particular detection method as a potential diagnostic method. The method according to the invention provides an additional new degree of freedom for assay development, since the synthesis process of the corresponding target sequence-specific probe is not necessarily complicated. Furthermore, the sensitivity of the assay according to the invention can be significantly increased, since additional nucleic acid sequences can be included in each reaction. Another advantage is that a suitable set of target sequence non-specific reporter molecules will always generate a population in a similar region of data space, thereby allowing conclusions to be made about activated reporters independent of the target sequence in the detection reaction, and therefore the same set can be used for different nucleic acid target sequence panels without time-consuming optimization.

[0034] In an embodiment of the method according to the invention, e. analysis of the signal, the signal intensity and / or the emission spectrum of the signal under a time point comprises displaying and / or analyzing the detected signal according to the signal intensity and / or the detection channel and / or the emission spectrum in a data space spanned by the evaluated detection channels.

[0035] In the context of the present invention, the data space is preferably generated by plots, diagrams or graphs of the different detection channels or by purely mathematical evaluation, comparison or contrast and / or algorithm-based evaluation, comparison or contrast and / or computer-based evaluation, comparison or contrast of the different detection channels, whereby from the fluorescent signals of each of the target sequence non-specific reporters used, data points in this 1-n dimensional data space (depending on the number of detection channels required for the evaluation) are generated and the data points can be grouped into clusters (groups, sets).

[0036] In the context of the present invention, the data space is preferably the space in which the displayed (detected signals) data is represented in a plot, diagram or graph, which spans the X-axis and Y-axis (2-dimensional plot), and optionally additionally spans the Z-axis in the case of a 3-dimensional graph, or further axes in the case of correspondingly higher dimensions. In an embodiment in which the detection channels used in the process are displayed on the axes of the graph, for example, the signals detected in the red detection channel are displayed on the X-axis and the signals detected in the green detection channel are displayed on the Y-axis, the data space can also be referred to as "detection channel spanned". The detected signals displayed in the data space are preferably unambiguously assignable to the target sequence, and may be preferably displayed in the form of a separate signal population. This is also preferably possible when multiple signals specific for different target sequences are detected in the same detection channel.

[0037] In an embodiment, the reporter molecules used simultaneously in the PCR reaction differ from each other by different fluorophore types (e.g., color, emission spectrum, and / or intensity), and / or by different quencher types, and / or by the number of labels on different reporter molecules. In an embodiment of single-color multiplexing according to the present invention, at least two target nucleic acids in the same detection channel are indirectly detected by reporter molecules with different or distinguishable signals. In an embodiment in which there are three or more target nucleic acids to be detected, the signal generated for the detection of the target nucleic acid can be divided between multiple detection channels (and labels of different reporter molecule types can be selected accordingly), if necessary, where it is preferred to indirectly detect at least two target nucleic acids in the same detection channel by reporter molecules with different or distinguishable signals.

[0038] In embodiments, at least a first nucleic acid target sequence and a second nucleic acid target sequence, or even at least the first target sequence, the second target sequence and a third target sequence, or even a fourth target sequence, a fifth target sequence, a sixth target sequence, a seventh target sequence, an eighth target sequence, a ninth target sequence, a tenth target sequence, an eleventh target sequence, a twelfth target sequence, a thirteenth target sequence, a fourteenth target sequence, a fifteenth target sequence, a sixteenth target sequence, a seventeenth target sequence, an eighteenth target sequence, a nineteenth target sequence, a twentieth target sequence, a twenty-first target sequence, or even more target sequences may be detected or the presence thereof may be detected.

[0039] Depending on the number of nucleic acid target sequences to be detected, at least a first mediator probe, a second mediator probe, a third mediator probe, a fourth mediator probe, a fifth mediator probe, a sixth mediator probe, a seventh mediator probe, an eighth mediator probe, a ninth mediator probe, a tenth mediator probe, an eleventh mediator probe, a twelfth mediator probe, a thirteenth mediator probe, a fourteenth mediator probe, a fifteenth mediator probe, a sixteenth mediator probe, a seventeenth mediator probe, an eighteenth mediator probe, a nineteenth mediator probe, a twentieth mediator probe, a twenty-first mediator probe or even more mediator probes are provided, each comprising an oligonucleotide comprising a mediator sequence and a probe sequence.

[0040] Thus, depending on the number of nucleic acid target sequences one wishes to detect, a first type, a second type, a third type, a fourth type, a fifth type, a sixth type, a seventh type, an eighth type, a ninth type, a tenth type, an eleventh type, a twelfth type, a thirteenth type, a fourteenth type, a fifteenth type, a sixteenth type, a seventeenth type, an eighteenth type, a nineteenth type, a twentieth type, a twenty-first type or even more types of target sequence-independent reporter molecules may be provided and used in the methods or kits according to the invention as described herein.

[0041] Those skilled in the art will understand how to combine target sequence independent reporter molecules according to the present invention and their label types to achieve detection of a desired target sequence according to the present invention.

[0042] In an embodiment, the label of the at least one target sequence independent reporter molecule comprises at least one label or at least two complementary oppositely oriented nucleobases each having at least two oppositely oriented bases offset by one base position from complementary base pairing each having at least one label.

[0043] The present invention allows for the simultaneous detection (multiplexing) of multiple target nucleic acids, where the number of simultaneously detected target nucleic acids can be greater than the number of detection channels available in the detection device.This is made possible by the fact that the reporter molecules used in each case contain labels that generate signals that can be distinguished from each other and can be represented in the analysis as separate (non-overlapping) signal populations in two- or three-dimensional data space, despite the simultaneous detection of the signals of multiple different reporter molecules in the same detection channel.In embodiments, this allows for the discrimination of signals that indicate the presence of different target nucleic acids, despite the detection in the same detection channel, and in certain embodiments, also allows for discrimination from background noise.

[0044] In an embodiment of the method according to the invention, in step d. or in step e., n different nucleic acid target sequences are indirectly detected by n different target sequence-independent reporter molecule types, and the detection of the signals generated in step d. is carried out in k detection channels, where n>k and n>2, At least two different target sequence-independent reporter molecule forms are detected in the same detection channel in step e. and / or displayed within the same region of the data space of the display under time point e.

[0045] By using the method according to the present invention, in an embodiment, it is possible to perform data classification in a higher dimensional data space (more than three dimensions, using four or more detection channels). In an embodiment, the method according to the present invention allows at least doubling the number of distinguishable target sequences in digital PCR compared to the available detection channels, provided that it is not essentially hindered by technical limitations on the device side, such as possible crosstalk between different detection channels (mutual interference of fluorescent signals; a fluorescent signal is erroneously associated with the signal of another fluorophore due to erroneous detection of fluorescence from an adjacent detection channel).

[0046] In embodiments, the signal of the reporter molecule label is suppressed, eliminated or quenched until the mediator sequence binds to the reporter molecule and is extended, preferably by a polymerase as part of a nucleic acid amplification reaction, e.g., in embodiments, spatial separation of at least one fluorophore from at least one quencher is achieved by the polymerase, such that a signal is generated that can be detected.

[0047] In an embodiment of the method according to the invention, the signal of the label of the target sequence independent reporter molecule is generated by cleavage and / or separation of the target sequence independent reporter molecule and / or by spatial separation of the at least one fluorophore and the at least one quencher.

[0048] In an embodiment, at least one target sequence-independent reporter molecule is an oligonucleotide or an oligonucleotide conjugate.

[0049] In an embodiment of the method according to the invention, the nucleic acid detection reaction in step d. comprises a DNA and / or cDNA amplification method.

[0050] In an embodiment of the method according to the present invention, the nucleic acid detection reaction under step d. is PCR, RT-PCR, RPA or LAMP, during the process of DNA amplification, the mediator sequence of the mediator probe bound to the target nucleic acid is released by the enzymatic activity of the biomolecule, and then the mediator probe binds to the target sequence-independent reporter molecule, resulting in the generation of a signal.

[0051] In an embodiment, the detection under step e. is performed as part of digital amplification and / or signal generation.

[0052] In an embodiment of the method according to the invention, the target sequence independent reporter molecule is a universal reporter and / or a modular reporter complex and at least one (respectively) released mediator sequence is a component of mediator-probe PCR or mediator-displacement LAMP.

[0053] In an embodiment, the nucleic acid target sequence is derived by conversion of DNA sequence information from another biomolecule.

[0054] In some embodiments, the presence and / or amount of an RNA sequence (one embodiment of a biomolecule) in a sample can be determined by transcribing the RNA sequence into cDNA by a reverse transcription reaction, which in embodiments can be amplified as part of a method according to the invention and indirectly detected by a target sequence-independent reporter molecule.

[0055] In an embodiment of the method according to the invention, the at least one label comprises a label selected from an electrochemically active label and / or a magnetic label.

[0056] In an embodiment of the method according to the invention, the signals are read at different temperatures.

[0057] In some preferred embodiments, the mediator probe comprises an oligonucleotide and a sequence-specific probe portion that binds to the target sequence and is protected at the 3' end. This protection at the 3' end may be a blocking group, e.g., a chemical blocking or protecting group, and in some embodiments comprises a chain of three carbon atoms. The protection of the mediator probe at the 3' end is preferably one that prevents (non-specific) extension of the sequence strand by a polymerase during an amplification reaction. In accordance with the present invention, in an embodiment, the mediator probe may 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 from (non-specific) polymerase extension by means other than a blocking group at the 3' end.

[0058] In other embodiments, the mediator probe does not contain a blocking group (protecting group) at the 3' end and is not protected from (non-specific) polymerase extension.

[0059] In embodiments, the mediator probe protected at the 3' end may comprise a "C3 spacer". Such a C3 spacer may in some embodiments be a chemical blocking group comprising a chain of three carbon atoms. This "C3 spacer" therefore preferably prevents (non-specific) polymerase extension of the mediator probe sequence chain. Those skilled in the art are familiar with typical and embodiment-dependent suitable blocking groups (protecting groups). Based on the disclosure of the present invention, those skilled in the art will know how to select suitable blocking groups (protecting groups) for routine application of the invention described herein.

[0060] Thus, the method according to the invention preferably allows direct end-point detection of n targets in k detection channels, where n>k and n≧2. In contrast to the prior art, the signal according to the invention is generated not by a fluorogenic probe, but by a target sequence-independent reporter molecule, which may also be described as a population-specific reporter, and preferably has a uniquely different intensity range. This ensures high flexibility in the experimental design process and allows the use of reporter molecules that utilize contact quenching for initial signal suppression. Furthermore, it allows controlled signal generation, resulting in more distinguishable and robust fluorescent signals that are particularly suitable for end-point detection. A preferred embodiment of the invention is based on a universal reporter structure, which is used in combination with a mediator probe for target sequence detection. One of the basic principles of the method according to the invention is preferably to detect n target nucleic acid sequences by n target-specific mediator probe types that activate n reporter molecule types, where each reporter molecule type is labeled with a unique fluorophore with defined optical properties (Figure 1). In the example of a dual reaction, two fluorophores are selected that fluoresce in the same wavelength range but with different intensities in the detection range. During dPCR amplification, the bound unlabeled mediator probe is cleaved by the polymerase, releasing the mediator sequence. The mediator activates the reporter molecule type, which then generates a fluorescent signal. The sum of all droplets or compartments with a signal of a certain reporter molecule type forms a population in a certain region of the data space, which corresponds to a unique fluorescent labeling. A reporter molecule type labeled with a fluorophore with a higher quantum yield generates a positive population with a higher fluorescence intensity (lower part of FIG. 1: higher intensity is shown as a larger circle), and a reporter molecule type labeled with a fluorophore with a lower quantum yield in the detection region generates a positive population with a lower fluorescence intensity (upper part of FIG. 1: lower intensity is shown as a smaller circle).Depending on their location in the data space, the formed populations can be assigned to the specific target DNA sequences that one wishes to detect in the sample. When two targets are present in one compartment, an additive effect of fluorescence intensity is assumed, which leads to the formation of a third population with a higher signal intensity. In further embodiments, a similar signal generation effect can be observed, for example, by using two different quencher types or two similar fluorophore labels for the reporter molecules in a dual reporter molecule-dPCR reaction. In the example of a quadruple reporter molecule dPCR assay carried out in two detection channels, the presence of multiple targets in one compartment leads to the formation of orthogonal populations. This means that by using four different reporter molecule types, up to 16 different populations can theoretically be detected and discriminated (e.g., FIG. 5). Without being bound by theory, it can be assumed that in digital assays, the distribution of target nucleic acids follows Poisson statistics. Thus, the generation of dual and multiple positive populations can depend on the concentration of the target nucleic acid as well as possible competitive effects caused by the detection of loci that share the same primer pair. Compared with the prior art high order multiplexing methods, the present method has the advantage that the concentration of the target sequence independent reporter molecule can be kept constant in different assays. This allows more freedom in the design of the assay and allows the fluorescent signal population to be optimized with high efficiency. In SNP detection, another advantage of combining a mediator probe with a target sequence independent reporter molecule comes into play. During the extension process, the bound mediator probe is preferably cleaved by the polymerase in a highly specific manner, cleaving the mediator portion and the probe portion. The cleavage site is preferably located between the first and second nucleotides of the probe portion of the mediator probe. Only when the cleavage is performed correctly can the released mediator bind sufficiently to the mediator binding site on the specific target sequence independent reporter molecule type to initiate signal generation.If proper cleavage does not occur, the extension process is preferably prevented in the target sequence-independent reporter molecule by the existing 3' overhang, and no signal is generated. Therefore, in the case of SNP detection, it is preferred that the first nucleotide is located at the 5' end of the sequence-specific probe portion of the mediator probe that spans the SNP position, so that it can activate a specific target sequence-independent reporter molecule type.

[0061] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: at least one first type of target sequence-independent reporter molecule comprising at least one label; Optionally, at least one second type of target sequence-independent reporter molecule comprising at least one label, which preferably emits a signal in the same detection channel or in a corresponding region of the data space of the at least one first type of target sequence-independent reporter molecule; Optionally, at least one first mediator probe, the mediator sequence having affinity for at least one first type of target sequence independent reporter molecule, the oligonucleotide sequence exhibiting affinity for a first nucleic acid target sequence; Optionally, at least one second mediator probe, in which the mediator sequence has affinity for at least one second type of target sequence-independent reporter molecule and the oligonucleotide sequence exhibits affinity for a second nucleic acid target sequence; Optionally, at least one buffering agent; Optionally, a polymerase; Optionally, a reverse transcriptase, Optionally, at least one PCR primer; The present invention relates to a kit comprising:

[0062] In one embodiment, a kit according to the invention is suitable or configured for carrying out a method according to the invention.

[0063] In an embodiment, a kit according to the present invention may comprise three or more different types of target sequence-independent reporter molecules, and may optionally comprise more mediator probes than the first mediator probe and the second mediator probe, wherein the mediator sequences of the mediator probes have affinity for the first type or the second type or the further type, respectively, of the target sequence-independent reporter molecules, and the oligonucleotide sequences of the mediator probes correspondingly have affinity for the first nucleic acid target sequence or the second nucleic acid target sequence or the further nucleic acid target sequence, respectively, and thus can specifically detect three or more different nucleic acid target sequences.

[0064] An embodiment in which one aspect of the invention is described may also be an embodiment of any of the other aspects of the invention. Thus, an embodiment in which a method according to the invention is described is also an embodiment of a kit according to the invention. Moreover, any embodiment described herein may include features of any other embodiment of the invention. The various aspects of the invention are unified by, benefit from, are based on, and / or relate to the unexpected advantageous effect of the method, namely the general surprising discovery of optimized simultaneous PCR detection by target sequence-independent reporter molecules of multiple target sequences.

[0065] The present invention is further illustrated by the following drawings, which are not intended to limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention presented to illustrate the invention described herein. [Brief description of the drawings]

[0066] [Figure 1]1 shows direct single-color multiplexing in digital PCR by using two target sequence-independent reporter molecules ("ZUR") of a first type (A) and a second type (B), each with different fluorophore labeling (shown as circles or triangles) with maximum emission in the red detection channel in a configuration that allows signal identification. The intensities of the different fluorescence signals characteristic of each nucleic acid target sequence are represented by circles of different sizes. By using target sequence-independent reporter molecules, in contrast to target sequence-specific Taqman probes, contact quenching (contact quenching of at least one fluorophore by at least one quencher) can be tuned more efficiently, thereby amplifying the difference in signal intensity of different fluorophores in the same detection channel. At least one quencher is located at the 5' end of the reporter molecule ("5'-quencher modification"). Each type of reporter molecule ("ZUR 1" under (A) or "ZUR 2" under (B)) is bound by a specific mediator sequence ("Mediator 1" under (A) or "Mediator 2" under (B)) that has previously been cleaved and / or released from the corresponding mediator probe ("Mediator Probe 1" under (A) or "Mediator Probe 2" under (B)), activating that reporter molecule. [Diagram 2] 1 is a graph showing the results of Example 3 by one-dimensional plots (1D plots). In Example 2, two nucleic acid target sequences were detected by (direct) single-color ("mediator probe") duplex PCR (digital PCR in the Stilla Naica system) using a first type and a second type of related specific target sequence-independent reporter molecule type. The two different reporter molecule types contain either the red fluorophore Cy5 or the red fluorophore Atto 647N as labels, which are characteristic of the respective nucleic acid target sequences, and are detected and analyzed in the red detection channel of the Stilla Naica system (digital PCR system). [Diagram 3]Graph showing the results of Example 3 by a two-dimensional (2D) plot. In Example 2, two nucleic acid target sequences were detected by (direct) single-color ("mediator probe") duplex PCR (digital PCR in Stilla Naica system) using a first type and a second type of related specific target sequence-independent reporter molecule type. The two different reporter molecule types contain either the red fluorophore Cy5 or the red fluorophore Atto 647N as labels, which are characteristic of the respective nucleic acid target sequences, and are detected in the red detection channel of the Stilla Naica system (digital PCR system). In this plot, the specific signal of the reporter complex / reporter molecule in the red channel ("red", Y-axis) is compared with the background signal (background noise) in the blue detection channel ("blue", X-axis) in a 2D data space. Units of the Y-axis: relative fluorescence units (RFU). [Figure 4] Graph showing the results of Example 3 by a three-dimensional (3D) plot. In Example 2, two nucleic acid target sequences were detected by (direct) single-color ("mediator probe") duplex PCR (digital PCR in the Stilla Naica system) using a first type and a second type of related specific target sequence-independent reporter molecule type. The two different reporter molecule types contain either the red fluorophore Cy5 or the red fluorophore Atto 647N as labels, which are characteristic of the respective nucleic acid target sequences, and are detected in the red detection channel of the Stilla Naica system (digital PCR system). In this plot, the specific signal of the reporter complex / reporter molecule in the red channel ("red", Y axis) is compared with the background signal (background noise) in the blue detection channel ("blue", X axis) and the signal of the negative control (not containing the target sequence) in the green detection channel ("green", Z axis) in the 3D data space. Axes units: relative fluorescence units (RFU). [Diagram 5]1 is a schematic diagram of the test design and reporter molecules used in Example 4. In Example 4, direct single-color multiplexing in two detection channels (e.g., red channel (X-axis) and green channel (Y-axis)) is performed in a digital PCR reaction using four different types of target sequence-independent reporter molecules (ZUR1 to ZUR4) with different fluorescent labels. A specific mediator sequence (mediator type 1 to type 4) binds to each of the four different types of target sequence-independent reporter molecules (ZUR1 to ZUR4), activating the reporter molecule. Here, the fluorescent signal generated and detected is specific to each nucleic acid target sequence. [Figure 6] Graph showing the results of Example 5. Two different target sequence-independent reporter molecule types were used in the red detection channel in a (direct) single-color multiplex PCR analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the signals indicating the presence of target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. The signals of the individual detections (after addition of only one DNA target sequence each) specific for target sequence 1 (X-axis) and target sequence 2 (X-axis) are shown in the center of the data space. The signal of the negative control (NTC; X-axis) is shown at the right edge of the data space. The signal intensity in the red detection channel ("Red") is shown on the Y-axis. Two types of target sequence-independent reporter molecules (ZUR05 and ZUR06) labeled with either Cy5 / BHQ-2 or BHQ-2 / Atto-647N were used for signal generation. Units of the Y-axis: relative fluorescence units (RFU). [Figure 7]Graph showing the results of this Example 5 for the use of two different reporter complexes / reporter molecules in the green detection channel in a (direct) single-color multiplexed analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the signals specific for target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. Two individual detections after the addition of only one DNA target sequence, respectively, for the detection of individual target sequence 1 (X-axis) or target sequence 2 (X-axis) are shown in the middle of the data space. The signal of the negative control (NTC; X-axis) is shown at the right edge of the data space. The signal intensity in the green detection channel is shown on the Y-axis. Two types of target sequence-independent reporter molecules (ZUR02 and ZUR04) labeled with either Dy530 / BMN-Q1 or BMN536 / BHQ-1 were used for signal generation. Units of the Y-axis: relative fluorescence units (RFU). [Figure 8] Graph showing the results of this Example 5 for the use of two different reporter complexes / reporter molecules in the blue detection channel in a (direct) single-color multiplexed analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the signals specific for target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. In the middle of the data space, the individual detections after addition of only one DNA target sequence are shown, showing the presence of the individual target sequence 1 (X-axis) or target sequence 2 (X-axis), respectively. The signal of the negative control (NTC; X-axis) is shown at the right edge of the data space. The signal intensity in the blue detection channel is shown on the Y-axis. Two types of target sequence-independent reporter molecules (ZUR07 and ZUR08), ​​labeled with either FAM / BHQ-1 or Atto488 / BMN-Q1, were used for signal generation. Units of the Y-axis: relative fluorescence units (RFU). [Figure 9]Graph showing the results of Example 6. Using (direct) single-color mediator probe multiplex PCR, samples were simultaneously analyzed for the presence of four target sequences in the green and blue detection channels. For this purpose, samples were amplified and analyzed in a corresponding digital PCR system (Stilla Naica system). In this "quadruplex mediator probe PCR", specific target sequence-independent reporter molecules were used for the target sequences I-KRAS G12A (KRAS gene mutation G12A), II-KRAS WT (KRAS wild-type gene), III-BRAF V600E (BRAF gene mutation V600E) and IV-BRAF WT (BRAF wild-type gene). When activated, the reporter molecule for the KRAS target gene variant generates a signal in the green detection channel (Y-axis), and when activated, the reporter molecule for the BRAF target gene variant generates a signal in the blue detection channel (X-axis). In the 2D plot (2D data space) shown, the signals of target sequence I and target sequence II detected individually in a distribution are shown in the upper left quadrant of the data space. The signals of target sequence III and target sequence IV detected individually in a distribution are shown in the lower right quadrant of the 2D data space. The signals of one KRAS target sequence and one BRAF target sequence detected simultaneously in one distribution (i.e., I+III, II+III, I+IV and II+IV) are shown in the upper right quadrant of the 2D data space. The signal intensity in the green detection channel is shown on the Y axis and the signal intensity in the blue detection channel is shown on the X axis. Axes units: relative fluorescence units (RFU). [Figure 10]Figure 1 shows the results of Example 6 in a three-dimensional (3D) plot. Using (direct) single-color mediator probe multiplex PCR, samples were analyzed simultaneously for the presence of six target sequences in the green, red and blue detection channels. For this purpose, samples were amplified and analyzed in a corresponding digital PCR system (Stilla Naica system). In this "6-plex mediator probe PCR", specific target sequence-independent reporter molecules (ZUR02, ZUR04-ZUR08) were used for the following target sequences: I-BRAF V600E (BRAF gene mutation V600E; ZUR07, labeled: FAM / BHQ-1), II-BRAF WT (BRAF wild-type gene; ZUR08, labeled: Atto 488 / BMN-W1), III-KRAS WT (KRAS wild-type gene; ZUR02, labeled: DY-530 / BMN-Q1), IV-KRAS G12A (KRAS gene mutation G12A; ZUR04, labeled: Cy5 / BHQ-1), V-KRAS G12D (KRAS gene mutation G12D; ZUR05, labeled: Cy5 / BHQ-2), VI-KRAS G12V (KRAS gene mutation G12V, ZUR06, label: Atto 647N / BHQ-2). When activated, the reporter molecules for the BRAF target gene variants BRAF V600E and WT generate signals in the blue detection channel (FAM or Atto 488 fluorophore), the reporter molecules for the KRAS WT and G12A target gene variants generate signals in the green detection channel (DY-530 or BMN-536 fluorophore), and the reporter molecules for the KRAS G12D and G12V target gene variants generate signals in the red detection channel (Cy5 and Atto-647N fluorophore). In the 3D plot (3D data space), the signals of target sequence I and target sequence II detected individually in the partition are shown on the X-axis. The signals of target sequence V and target sequence VI detected individually in the partition are shown on the Y-axis of the 3D data space. The signals of target sequence III and target sequence IV detected individually within the distribution are shown on the Z axis of the 3D data space.The signals of each KRAS or BRAF target sequence simultaneously detected within the distribution are also shown in the 3D data space. The signal intensity in the red detection channel is shown on the Y axis, the signal intensity in the blue detection channel is shown on the X axis, and the signal intensity in the green detection channel is shown on the Z axis. Axis units: relative fluorescence units (RFU). [Figure 11] One possibility of generating different signal intensities by using different labels for the reporter molecules results in an embodiment due to the physical molecular properties of the labels being amplified by contact quenching after activation of the target sequence non-specific reporter molecules. In both graphs, the X-axis shows the labels (or "markers") and the fixed dimension of the measured signal, such as wavelength, potential, frequency, etc. The variable dimension of the measured signal, in this case the signal intensity, is shown on the Y-axis. The points in each graph show the intersection of the signal intensity 1 and intensity 2 of fluorophore 1 or fluorophore 2, respectively, with the respective measured wavelengths. [Figure 12] Figure 9 shows the results of Example 9, where three different DNA target sequences were detected in one detection channel by three different target sequence non-specific reporter molecules, each with three different fluorescent labels, as part of a triplex mediator probe PCR using three different mediator probes in the infrared channel of Prism 6. In this "triple mediator probe PCR", the three target sequences I-KRAS G12D (KRAS single point mutation G12D), II KRAS WT (KRAS wild type genomic sequence) and III KRAS G12V (KRAS single point mutation G12V) were characterized by three target sequence non-specific reporter molecules with the fluorophore and quencher combinations Cy5.5+BHQ2 (I KRAS G12D), Dy636+BHQ2 (II KRAS WT) and Atto680+BHQ2 (III KRAS G12V). Thus, each of these target sequence non-specific reporter molecules generates a specific population in the data space that can be assigned to one of the three DNA target sequences. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The term "target sequence-independent reporter molecule" refers to a molecule or complex of at least one DNA oligonucleotide for generating a signal during PCR in the presence of a DNA target sequence. The target sequence-independent reporter molecule preferably comprises at least one label and at least one quencher.

[0068] In an embodiment, the target sequence independent reporter molecule comprises one or more mediator binding sites. Thus, the target sequence independent reporter molecule may 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. In a preferred embodiment, the target sequence independent reporter molecule comprises 1 to 10 mediator binding sites. The (activated) mediator or mediator sequence bound to the mediator binding site may, upon extension by a (PCR) polymerase, result in the activation or decomposition, digestion, (de)cleavage or release of one or more labels, e.g., quenchers and / or fluorophores, from one or more, preferably upstream (5'-end side) modifications located / positioned on the reporter molecule.

[0069] In the context of the present invention, mediator nucleic acid sequences may be referred to as "mediator probes" or "mediator probes". This always refers to a nucleic acid probe that transmits a signal between a target nucleic acid and a target sequence-independent reporter molecule that is homologous to the mediator probe. During a nucleic acid amplification reaction, such as a PCR or digital PCR reaction, the mediator probe bound to the target nucleic acid may be cleaved or digested by a polymerase moving forward on the target nucleic acid. The mediator sequence, or simply the "mediator", is then released, which may bind to the mediator binding site of the target sequence-independent reporter molecule. Now, when the polymerase also binds to the reporter molecule during the course of PCR and begins to extend the bound mediator sequence, the label is preferably activated. For example, the quencher is preferably separated from the fluorophore, resulting in the generation of a signal and / or a signal change. In other words, as used herein, "mediator" or "mediator sequence" refers to a nucleic acid oligonucleotide that can be extended by a polymerase along a target sequence-independent reporter molecule. A "mediator probe" refers to a nucleic acid oligonucleotide, preferably a DNA oligonucleotide, that binds to a target sequence during PCR in the presence of a DNA target sequence, is cleaved by the exonuclease activity of the polymerase, liberating a mediator sequence, which then binds to an associated target sequence-independent reporter molecule to which it has affinity, thereby establishing a link between the target sequence and the target sequence-independent reporter molecule.

[0070] In the context of the present invention, affinity for a nucleic acid target sequence refers to the physical attraction that two homologous nucleic acid sequences have for each other, which allows them to bind or hybridize to each other. In a preferred embodiment, for the affinity or successful hybridization of two homologous nucleic acid sequences to each other, there is complete homology between two nucleic acid sequences of a particular length, for example, between a probe sequence and a target nucleic acid sequence. In other embodiments, the homologous sequences may contain at least one mismatch with each other.

[0071] In the context of the present invention, the data space is preferably generated by plots, diagrams or graphs of different detection channels or by purely mathematical evaluation, comparison or contrast, algorithm-based evaluation, comparison or contrast and / or computer-based evaluation, comparison or contrast of different detection channels, where the fluorescence signals of each of the target sequence-independent reporter molecules used generate data points in this 1-n dimensional data space (depending on the number of detection channels required for evaluation), which data points can be grouped into clusters. Thus, in an embodiment, the "data space" can be the space in which the data displayed in the plot, diagram or graph is represented, which is preferably generated or "spread" by the X-axis and the Y-axis (2-dimensional plot), and optionally additionally the Z-axis in the case of a 3-dimensional graph, or additionally further axes in higher dimensions. In an embodiment in which the signals detected in each detection channel are displayed on the axes or plots of the graph, for example the signal of the red detection channel on the X-axis, the signal of the green detection channel on the Y-axis and the signal of the blue detection channel on the Z-axis of the plot, the data space can also be described as "spread by the detection channels". In a further embodiment, the data space is preferably "spread out" in terms of the detection channels analysed, generated by purely algorithm-based evaluation, comparison and / or contrast and / or computer-based evaluation, comparison and / or contrast of different detection channels and / or signals detected therein.

[0072] By using the method according to the present invention, in an embodiment, it is possible to perform data classification in a higher dimensional data space (more than three dimensions, using four or more detection channels). In an embodiment, the method according to the present invention can at least double the number of distinguishable target sequences in digital PCR compared to the available detection channels, provided that it is not essentially limited by the technical limitations of the device, such as possible crosstalk between different detection channels.

[0073] In the context of the present invention, a "label" may comprise one or more fluorophores and / or one or more quenchers. Thus, in the context of the present invention, a reporter molecule may have or comprise one or more fluorophores and / or quenchers. The proximity of a fluorophore to a quencher prevents the detection of the fluorescence of the fluorophore. In an embodiment, the proximity of the reporter and quencher is disrupted, for example, by the release of one or more fluorophores and / or quenchers, and in an embodiment, this disruption of proximity is achieved by the reporter molecule being partially degraded, for example at the 5' end, by hydrolysis by the 5' to 3' exonuclease activity of the PCR polymerase used in the amplification reaction. Thus, unquenched emission of fluorescence is possible, which can be detected after excitation with a laser. In a preferred embodiment, the target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher (which are thus preferably present in a "pair"), the quencher preferably suppressing the fluorophore signal until the mediator sequence binds to the reporter molecule, and is preferably extended by a polymerase as part of a nucleic acid amplification reaction. In an embodiment, for example, the polymerase provides spatial separation of the at least one fluorophore from the at least one quencher, resulting in the generation of a signal that can be detected. In an embodiment in which multiple quencher-fluorophore pairs are present in the target sequence-independent reporter molecule, the pairs may be positioned differently relative to each other. The target sequence independent reporter molecule may comprise one or more labels, for example there may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 labels, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or even 25 labels.In some embodiments, the target sequence independent reporter molecule comprises one, two, three or up to five labels, in other embodiments, the target sequence independent reporter molecule comprises one, two, three, four, five or even more labels.

[0074] For the purposes of the present invention, a "C3 spacer" is preferably a chemical blocking group (protecting group) and preferably comprises a chain of 3 carbon atoms. This blocking group (protecting group) preferably functions to prevent (non-specific) polymerase extension of the chain. The skilled person is familiar with typical and embodiment-dependent suitable C3 spacers / blocking groups (protecting groups) and, based on the present disclosure, the skilled person knows how to select a suitable C3 spacer / blocking group (protecting group) for routine application of the invention described herein.

[0075] According to the present invention, the signal-generating label is preferably a fluorophore or other dye capable of generating a detectable signal. In some embodiments, the signal-generating label is an electroactive label or a magnetic label. In the context of some embodiments, the terms "signal-generating label" and "label" are equivalent or interchangeable.

[0076] In the context of the present invention, biomolecules preferably comprise nucleic acids.

[0077] The term "nucleic acid" refers to a nucleic acid molecule, including, without limitation, DNA, ssDNA, dsDNA, RNA, mRNA, tRNA, lncRNA, ncRNA, microRNA, siRNA, rRNA, sgRNA, piRNA, rmRNA, snRNA, snoRNA, scaRNA, gRNA, or viral RNA. A nucleic acid sequence herein refers to a sequential arrangement of nucleotides, where the nucleotides are represented by the nucleobases guanine (G), adenine (A), cytosine (C), and thymine (T) in DNA and uracil (U) in RNA. A nucleic acid sequence herein may also refer to a 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. The nucleic acid sequence can be identified and characterized biochemically and bioinformatically by using DNA or RNA sequencing, or by specific detection by a complementary nucleic acid probe (e.g., in the present embodiment, by a mediator probe) as part of a detection reaction, for example, PCR, real-time PCR or digital PCR. Sequence analysis may include comparing the obtained nucleic acid sequence, or a detection signal specific to the nucleic acid sequence, with one or more reference nucleic acid sequences and / or with the detection signal of a housekeeping gene. The term nucleotide may be abbreviated as "nt". The term base pair (two nucleic acid bases bound to each other by hydrogen bonds) may be abbreviated as "bp".

[0078] In the context of the present invention, a "target sequence" (also referred to herein as "target") refers to any nucleic acid sequence of interest to be detected by the method according to the present invention. The target sequence may preferably be a DNA sequence, a cDNA sequence, a cfDNA sequence or an RNA sequence. The target sequence may be a portion of the target DNA or the entire nucleic acid sequence. The mediator probe preferably comprises a sequence that is fully or partially complementary to the nucleic acid sequence of the target sequence or a portion thereof. In some embodiments, the mediator probe sequence is 100%, 99%, 95%, 90% or 80% complementary to the target sequence. In some embodiments, the mediator probe may tolerate one or more mismatches to the target sequence and still bind to the target sequence. In other embodiments, the mediator probe binds to the target sequence only if it is 100% complementary to the target sequence.

[0079] The term "nucleic acid amplification reaction" refers to any process involving an enzymatic reaction that allows the amplification of nucleic acids. A preferred embodiment of the present invention relates to the polymerase chain reaction (PCR). "Polymerase chain reaction" ("PCR") is the gold standard method for rapidly generating millions to billions of copies (full or partial copies) of a given DNA sample, allowing very small amounts of DNA sample to be amplified to sufficiently large amounts. In PCR, a specific region of a DNA strand (the DNA target sequence) is amplified depending on where the primers used bind and initiate the amplification reaction. In almost all PCR applications, a thermostable DNA polymerase enzyme such as Taq polymerase is used.

[0080] In digital PCR (dPCR), the reaction mix and the nucleic acids contained therein (e.g., ctDNA or DNA) are divided into thousands of reaction spaces, which separates the target sequences from each other and facilitates their amplification and detection, especially for rare target sequences. A common method here is, for example, to dispense the reaction mix with the nucleic acid to be detected into a microfluidic drop in oil or into a fixed microcavity during PCR, whereby each formed unit forms a closed reaction space for amplification and detection. If a nucleic acid target sequence is present in the drop, it is amplified by PCR (or other amplification method). The individual amplification products are usually detected by sequence-specific fluorogenic nucleic acid probes, where a corresponding fluorescent signal is generated. In addition to absolute quantification, dPCR also has the advantage of reducing the influence of inhibitors. Since quantification in digital PCR is performed as an end-point analysis, "signal clusters" (also called populations) in the data space can be assigned to specific DNA or cDNA target sequences by the intensity of the fluorescent signal, which allows alternative types of data classification (Non-Patent Document 3, Non-Patent Document 4).

[0081] "Multiplexing" generally means that multiple target sequences are detected simultaneously in one reaction, where the reaction mixture does not need to be further divided for the detection of multiple target sequences. In "multiplex PCR", multiple individual PCR reactions for different DNA sequences or genes are combined under the same conditions to form a single reaction mixture. By being able to analyze multiple sequences simultaneously, information that would otherwise require many times more reagents and more time to perform can be obtained from a single PCR reaction. Thus, multiplexing reduces the cost and time required to perform PCR, since fewer reagents are used per experiment, experiments can be performed faster, and results can be analyzed more quickly. Since dividing the reaction mixture would cause the target sequence to be detected to be divided into a single detection reaction where it may not be detected, multiplex detection is more sensitive and therefore a very promising detection method, especially in tumor diagnosis. Using multiplex PCR can also improve accuracy compared to a single assay, since pipetting errors are also minimized with one reaction compared to multiple reactions.

[0082] Quantitative PCR ("qPCR") or "real-time PCR" is a specific form of PCR and is a standard method for detecting and quantifying specific target sequences in a sample in real time or quantifying gene expression levels. In qPCR, a fluorescently labeled probe or nucleic acid (e.g., a mediator probe) hybridizes in a PCR reaction and, in embodiments, once bound to a complementary sequence (e.g., a target sequence) during primer extension, is cleaved or digested by a PCR polymerase, where, in embodiments, the presence and amplification of the target sequence is monitored in real time after or during each PCR cycle. Real-time PCR allows the progress of an ongoing amplification reaction to be monitored on-the-fly (i.e., in real time). Thus, data is collected throughout the PCR reaction, rather than at an end point as with conventional PCR. Measuring the reaction rate at the early stages of PCR provides a significant advantage over conventional PCR detection. In embodiments of real-time PCR, the reaction is characterized by the time between cycles when target amplification is first detected, rather than by the amount of target accumulated after a fixed number of cycles as in conventional PCR. The higher the starting copy number of the nucleic acid target, the more likely it is that a significant increase in fluorescence will be observed. Real-time PCR allows analysis with optical signals that are used to detect specific PCR products (target sequences) using specific fluorescent dyes or fluorophores. Thus, an increase in DNA product during PCR leads to an increase in fluorescence intensity measured in each cycle. By using labels of different colors, fluorescent probes can be used in multiplex assays to monitor multiple target sequences.

[0083] Real-time qPCR relies on the relative amount of target nucleic acid determined in each amplification cycle, whereas in "digital PCR", it is possible to determine the absolute amount of target nucleic acid based on Poisson statistics used to calculate the amount of target nucleic acid after end-point PCR amplification. The pre-amplification steps are usually equivalent or similar in digital PCR and qPCR. However, in qPCR, it is preferred to pool all nucleic acid molecules and then amplify and analyze, whereas in digital PCR, it is preferred to separate nucleic acid molecules as far as possible into individual partitions (e.g., emulsion droplets, wells or gel beads), thereby allowing PCR to proceed as a single reaction in each partition (in the case of emulsion droplets, this reaction is often also called droplet PCR or digital droplet PCR), and allowing separate analysis of each partition. In digital PCR, the random partitioning of nucleic acid molecules into individual partitions occurs according to a Poisson distribution. Poisson statistics are then applied during the analysis of digital PCR to determine the average number of nucleic acid molecules per partition (none, one or more). Poisson statistical analysis of the number of positive and negative reactions provides rigorous absolute quantification of the target sequence.

[0084] Recombinase polymerase amplification ("RPA") is a method for amplifying DNA and is a variant of isothermal DNA amplification. RPA is usually performed using a recombinase (single-stranded binding protein) and a strand-displacing DNA polymerase, where the recombinase increases primer binding. When using a strand-displacing polymerase, the reaction can be performed at 37°C to 42°C or even at room temperature. Reverse transcription can be performed by adding a reverse transcriptase to the RPA reaction. In different variants of RPA, the resulting DNA can optionally be additionally quantified in the RPA reaction (similar to qPCR) and / or several DNA sequences can be amplified in parallel in a multiplex procedure.

[0085] The term "loop-mediated isothermal amplification" or "LAMP" refers to a method for amplifying DNA, and LAMP is a variation of isothermal DNA amplification. Isothermal amplification reactions are usually performed at a constant temperature. This characteristic distinguishes LAMP from PCR, in which the reaction is performed in a series of steps or cycles in which the temperature is changed. Strand-displacing DNA polymerases are usually used in LAMP. In a LAMP reaction, for example, 4 to 6 primers can be bound to 6 to 8 DNA sequences, and special primer design is required. In a LAMP reaction, reverse transcription can be performed by adding reverse transcriptase. Furthermore, the DNA generated in the LAMP reaction can be quantified. It is also possible to detect multiple DNA sequences in parallel in a multiplex LAMP reaction.

[0086] In the context of the present invention, a "signal change" refers to a change in a fluorescent signal. The signal change is preferably a significant, discriminable and / or characteristic change in a fluorescent signal that is clearly distinguishable or discriminated from a potential baseline or background signal or baseline or background noise. In the context of fluorescence detection, the skilled artisan recognizes that under some experimental conditions, a non-specific fluorescent base signal or baseline or background noise may occur due to a fluorophore. Thus, a signal change in the context of the present invention preferably refers to a significant, discriminable and / or characteristic change in a fluorescent signal, and not to a fluorescent base or background signal or baseline or background noise. In a preferred embodiment, the signal change may refer to an increase in fluorescence intensity, in other words, an increase in the fluorescent signal. In some embodiments, the signal change is a decrease in the fluorescent signal. The increase in the fluorescent signal is preferably due to the fact that the amplification reaction increases the number of target sequence amplification products and thus the activation of the associated signal generating complex. Thus, the resulting number of (de)cleavage, digestion and / or separation of each signal oligo from their binding sites on the relevant base strand increases, thereby liberating and / or separating at least one fluorophore from its quencher (i.e., the distance between the quencher and the fluorophore increases, such that the fluorescent signal is no longer quenched by the quencher). Thus, the increased (increased number) of liberated and / or unquenched fluorophores results in an increased fluorescent signal, which is specific for and indicative of the target sequence. Thus, in a PCR reaction, the more target sequence there is and the more mediator probes bound to it, the greater the increase in fluorescent signal. Preferably, the fluorescent 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.In the context of digital or "droplet" PCR, it is preferred that there is only one target sequence per reaction space (e.g., partition, emulsion droplet), so that the signal increases with the number of target sequence amplification products per reaction space until a signal plateau is reached that is predetermined by the maximum fluorescence intensity of the reporter molecule used for each detection. In a preferred embodiment, ideally, there is a uniform distribution of at most one target sequence per reaction space (e.g., partition, emulsion droplet) at the beginning of amplification, and thus, with similar amplification efficiency and similar maximum fluorescence intensity of the detection molecule in all reaction spaces (containing the target sequence), a specific signal is generated during reading by digital or "droplet" PCR that detects the same target sequence with comparable height / strength / intensity in different reaction spaces, which is preferably indicative of the presence and / or number of target sequence amplification products present in each reaction space. In an embodiment, the strength / intensity of each label, which is preferably specific to the target sequence, and the strength / intensity of the maximum achievable signal may depend on the number and / or type of label (e.g., fluorophore and / or quencher type) per signal oligo and signal complex.

[0087] A "fluorophore" (or fluorescent dye, similarly a chromophore) is a fluorescent chemical compound capable of re-emitting light upon optical excitation. Fluorophores for use as labels in the design of the labeled probes of the present invention are not claimed to be exhaustive and 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-acetic acid, 7-OH-4-CH3-coumarin-3-acetic acid, monobromobimane, pyrene trisulfonates such as Cascade Blue and monobromotrimethylammoniobimane, 7-NH2-4CH3-25-coumarin-3-acetic acid (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-R Including ho101, Cy5, Dy-636, Atto-647N, Cy5.5, Dy682, Atto-680, BMN-488, BMN-505, BMN-536, BMN-562.

[0088] "Quenching" refers to any process that reduces the fluorescence intensity of a given substance. Quenching is the basis of Förster resonance energy transfer (FRET) assays or static or contact quenching assays or a combination of both. FRET is a dynamic quenching mechanism since the energy transfer occurs while the donor is in an excited state. Contact quenching requires that the donor and quencher are in close proximity in the form of physical contact. A quencher is a molecule that quenches the fluorescence emitted from a fluorophore when it is excited by the light source of a PCR cycler or detection device. Quenchers used as labels in the design of the labeled signal oligos and / or base strands of the present invention are not claimed to be exhaustive and include 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. Those skilled in the art will know suitable reporter-quencher pairs and which to choose for a particular application. EXAMPLES

[0089] The invention will now be further described by the following examples, which are not intended to limit the scope of the invention but rather represent preferred embodiments of various aspects of the invention presented to illustrate the invention described herein.

[0090] method In the following examples, PCR amplification is carried out in the form of digital PCR. Digital PCR refers to a PCR amplification reaction in which preferably individual nucleic acid molecules are separated into individual partitions (e.g. emulsion droplets, wells or gel beads), so that PCR is carried out in each partition as an individual reaction (in the case of emulsion droplets, this reaction is often also called droplet PCR or digital droplet PCR), and each partition can be analyzed separately. This analysis is preferably carried out by fluorescence analysis of each individual partition (e.g. by laser excitation of a fluorophore and detection of the fluorescence emitted thereby). When a target sequence is located in a partition and detected by the method according to the invention, it is preferably indicated by a specific fluorescence signal.

[0091] In the examples described, the Naica system from Stilla Technologies was used. In a first step, droplets were generated and heat treated using a geode. The dPCR protocol used was 95°C for 5 min, followed by 45 cycles of 95°C for 15 s and 58°C for 60 s. Droplets were analyzed in examples 1 to 8 using a Prism 3 reader and CrystalMiner software (version 3.1.6.3) developed for this purpose, and in example 9 a Prism 6 reader was used.

[0092] For Examples 1-8, a single concentration of PerfeCTa™ MultiPlex qPCR ToughMix™ was used with 50 nM Alexa Fluor 488 as background fluorophore. In duplex and quadruple reactions, 0.5 μM forward primer and 0.25 μM reverse primer, 1.2 μM mediator probe and 0.6 μM target sequence independent reporter were used for each target sequence. For sexplex reactions, the concentration of target sequence independent reporter was reduced to 0.4 μM. In Example 9, a single concentration of naica™ Multiplex PCR MIX 10X was used, with 2 μM forward primer and 1 μM reverse primer overall, plus 1.2 μM mediator probe and 0.4 μM target sequence independent reporter for each target sequence.

[0093] In Example 10, the procedure can be similar to that of Example 9, with the mediator probe for ZUR used in Example 9 being adapted to an additional target sequence.

[0094] To prevent non-specific extension of the relevant oligonucleotide sequences, it is preferred to modify the 3'-end of the oligonucleotide sequence using chemical blocking groups. These are, for example, chains of three carbon atoms (C3 spacer) that are chemically bound to the oligonucleotide sequence and block its extension by the polymerase during the PCR reaction.

[0095] Sequence: Table 1: Primers [Table 1] SEQ ID: Sequence number Name:Name Sequence: Sequence (5'-3') Vonwarts: Forward Ruckwarts: Reverse

[0096] Table 2: Mediator probes. In the sequences, the probe region is shown in capital letters and the bases associated with the point mutation are shown in bold. The C3 spacer is preferably a chemical blocking group consisting of a chain of three carbon atoms that prevents non-specific polymerase extension of the strand. [Table 2] SEQ ID: Sequence number Name:Name Sequence: sequence (5'-3') Modification:3'Modification Spacer: C3 spacer

[0097] Table 3: Target sequence independent reporter molecules (fluorophore modification is located within the first 3' base of the stem-loop structure (shown as nucleotide 22 (7;6) or nucleotide 20 (8);6, 7 or 8); quencher is attached to the first base of the stem-loop structure on the 5' side (nucleotide 1)). The C3 spacer is preferably a chemical blocking group consisting of a chain of 3 carbon atoms that prevents non-specific polymerase extension of the chain. [Table 3] SEQ ID: Sequence number Name:Name Sequence: sequence (5'-3') Mod. Quencher: 5' modified quencher Modification:3'Modification Internal Modification Fluorophor: Internal modification with fluorophores Spacer: C3 spacer

[0098] Table 4: Target sequences (primer hybridization regions are underlined and point mutations / SNPs are shown in bold) [Table 4] SEQ ID: Sequence number Name:Name Sequence::Sequence(5'-3')

[0099] Example 1 In this embodiment, a signal is generated when the mediator probe binds to a nucleic acid target sequence and is digested or cleaved by a polymerase that amplifies the nucleic acid sequence as part of a PCR reaction. Thus, the mediator, or mediator sequence, is free to bind to a target sequence-independent reporter molecule. When the bound mediator is now recognized by the polymerase as a primer or initiator oligonucleotide for amplification during a PCR reaction, it is bound and extended by the polymerase, causing the quencher or fluorophore located "downstream" (downstream in the direction of amplification) of the mediator binding site to be separated or dislodged, resulting in the generation of a signal by the fluorophore that was previously quenched by contact quenching. This signal is characteristic of the nucleic acid target sequence to which the mediator probe was previously bound. The method according to the invention allows (direct) single-color multiplexing in digital PCR by using at least two target sequence-independent reporter molecules (see, for example, "target sequence-independent reporter" in FIG. 1), each with a different fluorophore modification (see, for example, FIG. 1, shown as a circle or triangle in the figure) with an emission maximum in one (e.g., red) detection channel in a configuration that allows signal identification. In FIG. 1, different signal intensities of fluorescence are represented by circles of different sizes. By using target sequence-independent reporter molecules, in contrast to the target sequence-specific Taqman probes of the prior art, contact quenching can be regulated more efficiently, thereby making it easier to amplify or model the difference in signal intensity of different fluorophores in the same detection channel.

[0100] In the prior art, for (direct) multiplexing, i.e. simultaneous detection of different target sequences in the analyzed sample and / or in the reaction, multiple optical channels, additional process steps or complex concentration adjustment or modification of reporter molecules are usually required. In contrast, the method according to the present invention allows the detection of different target sequences in a sample in the same detection channel, without being limited to the points listed above. The use of target sequence-independent reporter molecules also allows the optimized designs to be reused for different target sequences, making their application correspondingly more efficient. For example, different intensity combinations of the respective labels and / or different fluorophore colors, preferably different extended mediator sequences, thus allowing different detected target sequences to be coded and differentiated.

[0101] In an embodiment, multiple target sequences can be detected simultaneously with different fluorescence intensities and / or colors by using different target sequence-independent reporter molecules, each of which has a different mediator binding site with a fluorescent and / or quencher label with different signal intensities and / or colors / emission spectra. However, it is preferred to use target sequence-independent reporter molecules, of which at least two reporter molecules can always generate signals that can be measured / detected in the same detection channel.

[0102] The generation of signals with different signal intensities allows them to be distinguished within one fluorescent channel. These embodiments offer the important advantage of being able to distinguish different signal clusters, especially in digital PCR, and represent an improvement over the prior art.

[0103] Example 2 Another embodiment of (direct) single-color multiplexing according to the present invention is to use two target sequence-independent reporter molecules with different numbers of fluorophores and quenchers and with emission maxima in red detection channel.In this embodiment, different numbers of fluorophores generate different intensity fluorescent signals during digital PCR, thus allowing signal identification.By using target sequence-independent reporter molecules, as opposed to target sequence-specific Taqman probes, symmetric multiple modifications of fluorophores and quenchers can be used more efficiently, thereby amplifying the difference in signal intensity of different fluorophores in the same detection channel.

[0104] Example 3 In one sample analysis, (direct) single-color multiplexing was performed by using two target sequence-independent reporter molecules with different fluorophore labels in the red detection channel. Here, the two different target sequence-independent reporter molecules were labeled with either Cy5 or Atto-647N. The analysis included amplification reactions in the context of digital PCR (mediator probe PCR) in the Stilla Naica system, and the results obtained can be graphically displayed in a multi-dimensional data space.

[0105] In FIG. 2, the graphical evaluation and display of digital PCR results is shown in the form of a 1D (one-dimensional) plot.

[0106] In Figure 3, a graphical evaluation and display of digital PCR results is shown in the form of a 2D (two-dimensional) plot, where a specific signal of a reporter complex / reporter molecule in the red channel ("red", Y-axis) is compared to a background signal (background noise) in the blue detection channel ("blue", X-axis) in a two-dimensional data space. The two different signals of the reporter complex / reporter molecule can be easily distinguished from each other as separate signal populations in the two-dimensional data space, and also from the background noise (in the blue detection channel).

[0107] In Figure 4, the graphical evaluation and display of digital PCR results is shown in the form of a 3D (three-dimensional) plot. Here, in the three-dimensional data space, the specific signal of the reporter complex / reporter molecule in the red channel ("red", Y-axis) is compared with both the background signal in the blue detection channel ("blue", X-axis) and the signal of the negative control (sample without target gene) in the green channel ("green", Z-axis). In this example, the two different signals of the reporter complex / reporter molecule can be easily distinguished from each other as separate signal populations even in the three-dimensional data space, and can also be easily distinguished from the background noise and the signal of the negative control that was not detected in this example but potentially exists in other analyses.

[0108] Example 4 In this experiment, four different types of target sequence-independent reporter molecules (ZUR1-ZUR4) with different fluorescent labels are used to perform (direct) single-color multiplexing in two detection channels (e.g., red and green channels) in a digital PCR reaction using a mediator probe. In a graphical representation of the signals generated by the different types of reporter molecules and detected during a digital PCR reaction, these can be represented in the form of a signal population in a data space (the space generated by the X-axis and Y-axis or spanned by the X-axis and Y-axis and in which the data are graphed).

[0109] In Figure 5, a theoretical schematic diagram is shown displaying the analysis of signals from the examples using four different reporter molecules (ZUR1-ZUR4), where different fluorescent signal intensities are represented by circles of different sizes and positions in the data space. The data space in Figure 5 spans a space in which the intensities of signals detected in the red detection channel (ZUR4 and ZUR2) are shown on the X-axis and the intensities of signals detected in the green detection channel (ZUR3 and ZUR1) on the Y-axis. The signal populations in the data space can be unambiguously assigned to one of the four types of activated target sequence-independent reporter molecules and can therefore be efficiently used to detect different target sequence panels (sets or combinations of target sequences) as opposed to target sequence-specific reporter molecules (e.g., Taqman probes).

[0110] Example 5 In a further embodiment of the present invention, samples in the red, green and blue detection channels can be analyzed in parallel during (direct) single-color mediator probe multiplex PCR. For this purpose, samples were amplified and analyzed in a corresponding digital PCR system, such as the Stilla Naica system. For each detection channel, two, one and no DNA target sequences were added in a duplex reaction (a reaction mixture containing two different reporter molecule types, each specific for one of the two DNA target sequences), so that in each detection channel both target sequences could be detected simultaneously, or only one of the target sequences could be detected, or no target sequence could be detected.

[0111] In Figure 6 the results of this example are shown for the use of two different reporter complexes / reporter molecules in the red detection channel in a (direct) single-color multiplexed analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the signals characteristic of target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. In the middle of the data space, the signals of a reaction where a single target sequence was added (single reaction in a double reaction procedure) are shown, where these signals are specific for the individual target sequence 1 (X-axis) or target sequence 2 (X-axis). The signals measured for the negative control (NTC; X-axis) are shown at the right edge of the data space. The Y-axis shows the signal intensity detected in the red detection channel. Two types of target sequence-independent reporter molecules (ZUR05 and ZUR06), labeled with either Cy5 / BHQ-2 or BHQ-2 / Atto-647N, were used for signal generation.

[0112] The different signals of the reporter complexes / reporter molecules could be easily distinguished from each other as separate signal populations in the two-dimensional data space, thus allowing specific detection of both target sequences in each case.

[0113] In Figure 7 the results of this example are shown for the use of two different reporter complexes / reporter molecules in the green detection channel in a (direct) single-color multiplexed analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the detected signals specific for target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. Two individual detections after adding only one DNA target sequence for the individual detection of target sequence 1 (X-axis) and target sequence 2 (X-axis), respectively, are shown in the middle of the data space. A negative control (NTC; X-axis) is shown at the right edge of the data space. The signal intensity in the green detection channel is shown on the Y-axis. Two types of target sequence-independent reporter molecules (ZUR02 and ZUR04) labeled with either Dy530 and BMN-Q1 or BMN536 and BHQ-1 were used for signal generation.

[0114] The different signals of the reporter complexes / reporter molecules could be easily distinguished from each other as separate signal populations in the two-dimensional data space, thus allowing specific detection of both target sequences in each case.

[0115] In Figure 8 the results of this example are shown for the use of two different reporter complexes / reporter molecules in the blue detection channel in a (direct) single-color multiplexed analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, where the signals specific for target sequence 1 and target sequence 2 (X-axis) are shown at the left edge of the data space. Two individual detections after the addition of only one DNA target sequence, respectively, showing the presence of the individual target sequence 1 (X-axis) or target sequence 2 (X-axis) are shown in the middle of the data space. The signal of the negative control (NTC; X-axis) is shown at the right edge of the data space. The signal intensity in the blue detection channel is shown on the Y-axis. Two types of target sequence-independent reporter molecules (ZUR07 and ZUR08) labeled with either FAM / BHQ-1 or Atto488 / BMN-Q1 were used for signal generation.

[0116] The different signals of the reporter complexes / reporter molecules could be easily distinguished from each other as separate signal populations in the two-dimensional data space, thus allowing specific detection of both target sequences in each case.

[0117] Example 6 In a further embodiment of the present invention, in a (direct) single-color mediator probe multiplex PCR, samples in the green detection channel and the blue detection channel could be simultaneously analyzed for the presence of four target sequences, respectively. For this purpose, the samples were amplified and analyzed in a corresponding digital PCR system, such as the Stilla Naica system. In this "quadruplex mediator probe PCR", four target sequence-independent reporter molecules and four target sequence-specific mediator probes were prepared to detect the DNA target sequences I-KRAS G12A (KRAS gene mutation G12A), II-KRAS WT (KRAS wild-type gene), III-BRAF V600E (BRAF gene mutation V600E) and IV-BRAF WT (BRAF wild-type gene). Reporter molecules for KRAS target gene variants (different BRAF or KRAS single nucleotide polymorphisms (SNPs) or wild-type (WT) variants) generated a signal in the green detection channel when activated (BMN536 and Dy-530 fluorophores), and reporter molecules for BRAF target gene variants generated a signal in the blue detection channel when activated (FAM and Atto 488 fluorophores).

[0118] The results of this SNP analysis are shown in Figure 9, where the signals of target sequence I and target sequence II detected individually in the distribution are shown in the upper left quadrant of the data space. The signals of target sequence III and target sequence IV detected individually in the distribution are shown in the lower right quadrant of the data space. The signals of one KRAS target sequence and one BRAF target sequence detected simultaneously in one distribution (i.e., I+III, II+III, I+IV, and II+IV) are shown in the upper right quadrant of the data space. The signal intensity in the green detection channel (BMN536 and Dy-530 fluorophores) is shown on the Y axis, and the signal intensity in the blue detection channel (FAM and Atto 488 fluorophores) is shown on the X axis.

[0119] As can be seen in FIG. 9, multiple signal clusters can be detected per detection channel and assigned to activated target sequence-independent reporter molecules and therefore to the corresponding target sequences.

[0120] Example 7 In a further embodiment of the present invention, samples could be analyzed for the presence of six target sequences simultaneously in the green, red and blue detection channels during (direct) single-color mediator probe multiplex PCR. For this purpose, samples were amplified and analyzed in a corresponding digital PCR system, such as the Stilla Naica system. In this "six-plex mediator probe PCR", six target sequence-independent reporter molecules and six target sequence-specific mediator probes were prepared to analyze the target sequences I-BRAF V600E (BRAF gene mutation V600E), II-BRAF WT (BRAF wild-type gene), III-KRAS WT (KRAS wild-type gene), IV-KRAS G12A (KRAS gene mutation G12A), V-KRAS G12D (KRAS gene mutation G12D), and VI-KRAS G12V (KRAS gene mutation G12V). The reporter molecules for the BRAF target gene variants BRAF V600E and WT generated a signal in the blue detection channel when activated (FAM or Atto 488 fluorophore), the reporter molecules for the KRAS WT and G12A target gene variants generated a signal in the green detection channel when activated (DY-530 or BMN-536 fluorophore), and the reporter molecules for the KRAS G12D and G12V target gene variants generated a signal in the red detection channel when activated (Cy5 and Atto-647N fluorophores).

[0121] The results of this analysis are shown in Figure 10, where the signals of target sequences I and II detected individually in the distribution are shown on the X-axis of the data space. The signals of target sequences V and VI detected individually in the distribution are shown on the Y-axis of the data space. The signals of target sequences III and IV detected individually in the distribution are shown on the Z-axis of the data space. The signals of each KRAS or BRAF target sequence simultaneously detected in the distribution are also shown in the data space. The signal intensity in the red detection channel is shown on the Y-axis, the signal intensity in the blue detection channel is shown on the X-axis, and the signal intensity in the green detection channel is shown on the Z-axis.

[0122] As can be seen in FIG. 10, multiple signal clusters can be detected per detection channel and assigned to activated target sequence-independent reporter molecules and therefore to the corresponding target sequences.

[0123] Example 8 Using the described method, it is also possible to perform data classification in a higher dimensional data space (more than three dimensions, using four or more detection channels). Using the described method, the number of distinguishable target sequences in digital PCR can theoretically be at least twice the number of available detection channels, provided that it is not essentially limited by technical limitations of the device, such as crosstalk between different detection channels.

[0124] [Table 5] Number of detection channels on the device side: Number of detection channels on the device side Theoretical minimum number of differentiable target sequences

[0125] Those skilled in the art are familiar with corresponding methods for classifying higher dimensional data structures.

[0126] Example 9 In further examples, the described method can be used to increase the multiplexing capacity by more than 2-fold for each detection channel. Thus, the multiplexing capacity can be increased by at least 3-fold or even more per detection channel. This is made possible by using at least three reporter molecules in the detection channels, which generate at least three distinct populations in the data space, each of which is assigned specifically to each reporter. This allows the detection of up to three target sequences in one detection channel, tripling the detection channel capacity of the device, provided that it is not essentially limited by technical limitations of the device, such as crosstalk between different detection channels. The results of such a doubling of the detection channels in the Stilla Prism-6 are shown in FIG. 12. Here, the presence of three target sequences was simultaneously analyzed in the infrared detection channel of the Stilla Prism-6. For this purpose, the samples were amplified and analyzed in a corresponding digital PCR system, such as the Stilla Naica system. In this "triple mediator probe PCR", three target sequence-specific mediator probes were prepared to detect three target sequences, I-KRAS G12D (KRAS single point mutation G12D), II KRAS WT (KRAS wild type genomic sequence) and III KRAS G12V (KRAS single point mutation G12V), using three target sequence-nonspecific reporter molecules with fluorophore and quencher combinations Cy5.5+BHQ2 (I KRAS G12D), Dy636+BHQ2 (II KRAS WT) and Atto680+BHQ2 (III KRAS G12V). The particular advantage of this method over the prior art is that signal generation using target sequence-nonspecific reporter molecules makes signal generation very robust and populations are generated at comparable distances from each other in data space across multiple independent experiments, thus simplifying data analysis. This provides a greater advantage in signal optimization required for such population separation.

[0127] Example 10 In a further embodiment, due to the separation of the detection of one or more different DNA target sequences by the combination of cleavage of one or more different mediator probes and release of one or more different mediator sequences, and the signal generation by extension of one or more mediator sequences on one or more different target sequence-independent reporter molecules, one set of target sequence-independent reporter molecules can be used to detect different panels of DNA target sequences. In this case, the target sequence-independent reporter molecules remain constant, and the mediator probes and / or primers are replaced to accommodate different DNA target sequences. In this way, the efficiency of the optimization of the fluorescent signal of the target sequence-independent reporter is much increased compared to the prior art, since these optimizations only need to be performed once for each detection device (e.g., digital PCR thermal cycler). This means that the signal optimization can be used to generate many different highly discriminable fluorescent signal populations in the data space more efficiently than before, since this optimization only needs to be performed once and can then be moved to other DNA target sequence panels. The corresponding optimization process is therefore more efficient, is generally known to those skilled in the art and is described in the specialized literature (Non-Patent Document 11). This means that due to the optimization of the signal, an assay using direct single-color multiplexing can achieve better performance in terms of sensitivity, precision or specificity with less effort than prior art assays that need to optimize the signal again for each new target sequence. Compared to the prior art, this has the particular advantage that a signal can always be unambiguously assigned to the same reporter only by its position in the data space, and there is no need to correlate this position in the data space with other information, such as temperature or the position of other signal populations in the data space, to determine the activation of each target sequence non-specific reporter.

[0128] References Faltin, Bernd; Wadle, Simon; Roth, Guenter; Zengerle, Roland; Stetten, Felix von (2012): Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters. In: Clinical chemistry 58 (11), pp. 1546-1556. DOI: 10.1373 / clinchem.2012.186734. Garcia-Murillas, Isaac; Schiavon, Gaia; Weigelt, Britta; Ng, Charlotte; Hrebien, Sarah; Cutts, Rosalind J. et al. (2015): Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. In: Science translational medicine 7 (302), 302ra133. DOI: 10.1126 / scitranslmed.aab0021. Hughesman, Curtis B.; Lu, X. J. David; Liu, Kelly Y. P.; Zhu, Yuqi; Poh, Catherine F.; Haynes, Charles (2016): A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens. In: PloS one 11 (8), e0161274. DOI: 10.1371 / journal.pone.0161274. Kipf, Elena; Schlenker, Franziska; Borst, Nadine; Fillies, Marion; Kirschner-Schwabe, Renate; Zengerle, Roland et al. (2022): Advanced Minimal Residual Disease Monitoring for Acute Lym-phoblastic Leukemia with Multiplex Mediator Probe PCR. In: The Journal of molecular diagnostics : JMD 24 (1), pp. 57-68. DOI: 10.1016 / j.jmoldx.2021.10.001. Lehnert, Michael; Kipf, Elena; Schlenker, Franziska; Borst, Nadine; Zengerle, Roland; Stetten, Felix von (2018): Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides. In: Anal. Methods 10 (28), pp. 3444-3454. DOI: 10.1039 / C8AY00812D. Milbury, Coren A.; Zhong, Qun; Lin, Jesse; Williams, Miguel; Olson, Jeff; Link, Darren R.; Hutchison, Brian (2014): Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. In: Biomolecular detection and quantification 1 (1), pp. 8-22. DOI: 10.1016 / j.bdq.2014.08.001. Pecoraro S., Berben G., Burns M., Corbisier P., De Giacomo M., De Loose M., Dagand E., Dobnik D., Eriksson R., Holst-Jensen A., Kagkli D. M., Kreysa J., Lievens A., Maede D., Mazzara M., Paterno A., Peterseil V., Savini C., Sovova T., Sowa S., Spilsberg B.: Overview and recommendations for the application of digital PCR. JRC Technical Report. Luxembourg: Publications Office of the European Union, 2019 (2019). Available online at https: / / publications.jrc.ec.europa.eu / repository / handle / JRC115736. Schlenker, Franziska; Kipf, Elena; Borst, Nadine; Hutzenlaub, Tobias; Zengerle, Roland; Stetten, Felix von; Juelg, Peter (2021a): Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations. In: Analytical chemistry 93 (30), pp. 10538-10545. DOI: 10.1021 / acs.analchem.1c01488. Schlenker, Franziska; Kipf, Elena; Deuter, Max; Hoeffkes, Inga; Lehnert, Michael; Zengerle, Roland et al. (2021b): Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA. In: Cancers 13 (22). DOI: 10.3390 / cancers13225742. Schuler, Friedrich; Trotter, Martin; Zengerle, Roland; Stetten, Felix von (2016): Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes. In: Analytical chemistry 88 (5), pp. 2590-2595. DOI: 10.1021 / acs.analchem.5b02960. Whale, Alexandra S.; Huggett, Jim F.; Tzonev, Svilen (2016): Fundamentals of multiplexing with digital PCR. In: Biomolecular detection and quantification 10, pp. 15-23. DOI: 10.1016 / j.bdq.2016.05.002. Corne, Julien; Le Du, Fanny; Quillien, Veronique; Godey, Florence; Robert, Lucie; Bourien, He-loiese et al. (2021): Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients. In: Scientific reports 11 (1), p. 17316. DOI: 10.1038 / s41598-021-96644-6. Faltin, Bernd; Wadle, Simon; Roth, Guenter; Zengerle, Roland; Stetten, Felix von (2012): Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters. In: Clinical chemistry 58 (11), pp. 1546-1556. DOI: 10.1373 / clinchem.2012.186734. Garcia-Murillas, Isaac; Schiavon, Gaia; Weigelt, Britta; Ng, Charlotte; Hrebien, Sarah; Cutts, Rosalind J. et al. (2015): Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. In: Science translational medicine 7 (302), 302ra133. DOI: 10.1126 / scitranslmed.aab0021. Hughesman, Curtis B.; Lu, X. J. David; Liu, Kelly Y. P.; Zhu, Yuqi; Poh, Catherine F.; Haynes, Charles (2016): A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens. In: PloS one 11 (8), e0161274. DOI: 10.1371 / journal.pone.0161274. Kipf, Elena; Schlenker, Franziska; Borst, Nadine; Fillies, Marion; Kirschner-Schwabe, Renate; Zengerle, Roland et al. (2022): Advanced Minimal Residual Disease Monitoring for Acute Lym-phoblastic Leukemia with Multiplex Mediator Probe PCR. In: The Journal of molecular diagnostics : JMD 24 (1), pp. 57-68. DOI: 10.1016 / j.jmoldx.2021.10.001. Lehnert, Michael; Kipf, Elena; Schlenker, Franziska; Borst, Nadine; Zengerle, Roland; Stetten, Felix von (2018): Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides. In: Anal. Methods 10 (28), pp. 3444-3454. DOI: 10.1039 / C8AY00812D. Madic, Jordan; Jovelet, Cecile; Lopez, Julien; Andre, Barbara; Fatien, Jean; Miran, Isabelle et al. (2018): EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung can-cer revealed by six-color crystal digital PCR. In: Oncotarget 9 (100), pp. 37393-37406. DOI: 10.18632 / oncotarget.26446. Milbury, Coren A.; Zhong, Qun; Lin, Jesse; Williams, Miguel; Olson, Jeff; Link, Darren R.; Hutchison, Brian (2014): Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. In: Biomolecular detection and quantification 1 (1), pp. 8-22. DOI: 10.1016 / j.bdq.2014.08.001. Pecoraro S., Berben G., Burns M., Corbisier P., De Giacomo M., De Loose M., Dagand E., Dob-nik D., Eriksson R., Holst-Jensen A., Kagkli D. M., Kreysa J., Lievens A., Maede D., Mazzara M., Paterno A., Peterseil V., Savini C., Sovova T., Sowa S., Spilsberg B.: Overview and recommendations for the application of digital PCR. JRC Technical Report. Luxembourg: Publications Office of the European Union, 2019 (2019). Available online at https: / / publications.jrc.ec.europa.eu / repository / handle / JRC115736. Schlenker, Franziska; Kipf, Elena; Borst, Nadine; Hutzenlaub, Tobias; Zengerle, Roland; Stetten, Felix von; Juelg, Peter (2021a): Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations. In: Analytical chemistry 93 (30), pp. 10538-10545. DOI: 10.1021 / acs.analchem.1c01488. Schlenker, Franziska; Kipf, Elena; Deuter, Max; Hoeffkes, Inga; Lehnert, Michael; Zengerle, Roland et al. (2021b): Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA. In: Cancers 13 (22). DOI: 10.3390 / cancers13225742. Schuler, Friedrich; Trotter, Martin; Zengerle, Roland; Stetten, Felix von (2016): Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes. In: Analytical chemistry 88 (5), pp. 2590-2595. DOI: 10.1021 / acs.analchem.5b02960. Stilla Technologies: High multiplex, ultrasensitive EGFR detection using the EGFR 6-color Crystal Digital PCR TM kit. Stilla Technologies. Available online at https: / / www.stillatechnologies.com / wp-content / uploads / 2021 / 12 / StillaTechnologies-6-color-EGFR-kit-App-Note.pdf. Whale, Alexandra S.; Huggett, Jim F.; Tzonev, Svilen (2016): Fundamentals of multiplexing with digital PCR. In: Biomolecular detection and quantification 10, pp. 15-23. DOI: 10.1016 / j.bdq.2016.05.002. [Explanation of symbols]

[0129] Drawing translation Figure 1 DNA detection Signal generation Polymerase Primer Mediator probe Target DNA Mediator 5'-Quencher modification Target sequence-independent reporter Red fluorophore Type AA Type BB Figure 2 Red Droplet Index Droplet Index Figure 3 Red Blue Figure 4 Green Blue Red Figure 5 Green fluorescence channel-Intensity Red fluorescence channel-Intensity Green Red Mediator type 1 and 3 Mediator type 1-3 Mediator type 1, 3, 4 Mediator type 1-4 Mediator type 3 Mediator type 3 and 2 Mediator type 3 and 4 Mediator type 2-4 Mediator type 1 Mediator type 1 and 2 Mediator type 1 and 4 Mediator type 1, 2, 4 Mediator type 2 Mediator type 4 Mediator type 2 and 4 Non-activated ZUR R Non-activated ZUR R Figure 6 Red Target sequence Figure 7 Green Target sequence Figure 8 Blue Target sequence Figure 9 Green Blue Populations of DNA target sequences detected by the respective ZUR: ZUR07 with FAM and BHQ-1 ZUR08 with Atto 488 and BMN-Q1 ZUR02 with DY-530 and BMN-Q1 ZUR04 with BMN536 and BHQ-1 Figure 10 Green Blue Red Populations of DNA target sequences detected by the respective ZUR: Figure 11 Variable dimension of the measured signal: Signal strength Fixed dimension of the label and the measured signal e.g. wavelength, potential, frequency, ... Fluorophore Measuring wavelength Signal strength Fixed dimension of the measured signal e.g. wavelength, potential, frequency, ... Figure 12 INFRA-RED Infrared Target sequence I, II and III

Claims

1. 1. A method for detecting at least two nucleic acid target sequences in the same detection channel by at least two target sequence-independent reporter molecules, comprising: a. providing at least a first nucleic acid target sequence and a second nucleic acid target sequence; b. providing at least a first mediator probe and a second mediator probe, each comprising an oligonucleotide; the first mediator probe oligonucleotide comprises a mediator sequence and a probe sequence, the mediator sequence having affinity for a first type of target sequence-independent reporter molecule, and the probe sequence exhibiting affinity for a first nucleic acid target sequence; the second mediator probe oligonucleotide comprises a mediator sequence and a probe sequence, the mediator sequence having affinity for a second type of target sequence-independent reporter molecule, and the probe sequence exhibiting affinity for a second nucleic acid target sequence; the at least first mediator probe and the second mediator probe do not have a signal-generating label; At least one label of the target sequence-independent reporter molecule is at least one fluorophore and / or at least one quencher; The process and c. providing at least two target sequence-independent reporter molecules of at least a first type and a second type, each of which comprises at least one label having a measurable signal in the same detection channel and a nucleic acid sequence having a specific affinity for at least one mediator sequence, each type of said at least two target sequence-independent reporter molecules is characterized by at least one label such that it has a signal intensity distinguishable from the signal intensities of the labels of all other target sequence-independent reporter molecule types and can be directly assigned to a respective nucleic acid target sequence; d. performing a nucleic acid detection reaction, wherein upon binding of the probe sequence of the at least first mediator probe to the at least first nucleic acid target sequence, at least one mediator sequence of the at least first mediator probe is released; the at least one released mediator sequence binds to at least one first type of target sequence-independent reporter molecule, and the at least one label is assigned to each target sequence-independent reporter molecule, generating a signal that is characteristic due to signal intensity and / or emission spectrum for the first nucleic acid target sequence bound by at least one probe sequence of the at least one first mediator probe; e. detecting the signal generated in step d., comprising detecting the signal in a detection channel and / or analyzing the signal, its intensity and / or its emission spectrum; A method comprising:

2. e. The method of claim 1, wherein the analysis of the signal, signal intensity and / or emission spectrum of the signal at a time point includes displaying and / or analyzing the detected signal depending on the signal intensity and / or detection channel and / or emission spectrum in the data space spanned by the evaluated detection channel.

3. 3. The method of claim 1, wherein the different target sequence-independent reporter molecule types differ in the signal intensity and / or emission spectrum of at least one label thereof.

4. 3. The method of claim 1 or 2, wherein at least one label of the target sequence-independent reporter molecule comprises at least two fluorophores and / or two quenchers having the same or different emission spectra and / or the same or different signal intensities.

5. At least one label of the target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher, or 3. The method of claim 1, wherein at least one label of the target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher, and contact quenching occurs between the at least one fluorophore and the at least one quencher unless a mediator sequence binds to the corresponding target sequence-independent reporter molecule or unless the bound mediator sequence is extended during the nucleic acid detection reaction.

6. 3. The method of claim 1 or 2, wherein the label of the at least one target sequence-independent reporter molecule comprises at least one label or at least two complementary opposite-oriented nucleobases, each having at least two opposite-oriented bases offset by one base position from complementary base pairing, each having at least one label.

7. In step d. or step e., n different nucleic acid target sequences are indirectly detected by n different target sequence-independent reporter molecule types, and the detection of the signals generated in step d. is carried out in k detection channels; n>k and n≧2, 3. The method of claim 1, wherein at least two different target sequence-independent reporter molecule types are detected in the same detection channel in step e. and / or displayed in the same region of the data space of the display under step e.

8. 3. The method of claim 1, wherein the signal of the label of the target sequence-independent reporter molecule is generated by cleavage and / or separation of the target sequence-independent reporter molecule and / or by spatial separation of the at least one fluorophore and the at least one quencher.

9. The method of claim 1 or 2, wherein the target sequence-independent reporter molecule is an oligonucleotide.

10. The method of claim 1 or 2, wherein the nucleic acid detection reaction in step d. comprises a DNA and / or cDNA amplification method.

11. 11. The method according to claim 10, wherein the nucleic acid detection reaction in step d is PCR, RT-PCR, RPA, or LAMP, and during the DNA amplification process, the mediator sequence of the mediator probe bound to the target nucleic acid is released by the enzymatic activity of a biomolecule, and then the mediator probe binds to a target sequence-independent reporter molecule, resulting in the generation of a signal.

12. 11. The method of claim 10, wherein the detection in step e. is carried out in the context of digital amplification and / or signal generation.

13. 3. The method of claim 1 or 2, wherein the target sequence-independent reporter molecule is a universal reporter and / or a modular reporter complex, and the at least one released mediator sequence is a component of mediator-probe PCR or mediator-displacement LAMP.

14. 3. The method of claim 1 or 2, wherein at least one nucleic acid target sequence is generated by conversion of DNA sequence information from another biomolecule.

15. A kit for carrying out the method according to claim 1 or 2, comprising: at least one first type of target sequence-independent reporter molecule comprising at least one label; at least one second type of target sequence-independent reporter molecule comprising at least one label; at least one first mediator probe, the mediator sequence having affinity for said at least one first type of target sequence-independent reporter molecule and the oligonucleotide sequence exhibiting affinity for a first nucleic acid target sequence; at least one second mediator probe, the mediator sequence having affinity for said at least one second type of target sequence-independent reporter molecule and the oligonucleotide sequence exhibiting affinity for a second nucleic acid target sequence; It may or may not contain at least one element selected from the following: at least one buffer; - polymerase, - reverse transcriptase, - at least one PCR primer, Includes a kit.

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