PCR multiplexing through target sequence-independent reporter molecules with distinguishable signal strengths

Abstract

The invention relates to a method for specific detection of at least two nucleic acid target sequences through at least two target sequence-independent reporter molecules in the same detection channel, wherein at least two mediator probes, comprising at least one probe sequence and one mediator sequence, and at least two target sequence-independent reporter molecules of a first and second type, each comprising at least one label with a respective signal intensity maximum in the same detection channel, are employed and wherein the at least two nucleic acid target sequences are detected by means of a nucleic acid detection reaction due to a signal intensity and / or emission spectrum characteristic therefor. The invention also relates to a kit for performing the method.

Description

[0001] PCR multiplexing by target-independent reporter molecules with distinguishable signal intensities

[0002] TECHNICAL BACKGROUND

[0003] Precise and sensitive nucleic acid detection methods, as a further development of real-time PCR, especially digital PCR, have revolutionized molecular diagnostics in recent years, as they are superior to standard PCR in the detection of rare gene targets (Garcia-Murillas et al. 2015; Milbury et al. 2014). In digital PCR (dPCR), the reaction mix and the nucleic acids it contains (e.g., ctDNA or DNA) are divided into thousands of reaction chambers, which separates the target sequences and facilitates their amplification and detection, especially of rare target sequences. A common method is the microfluidic dropletization of the reaction mix containing the nucleic acid to be detected in oil or the division into solid microcavities, whereby each formed unit forms a closed reaction chamber for amplification and detection, for example, during PCR.If a nucleic acid target sequence is present in a droplet, it is amplified by PCR (or other amplification methods). The individual amplificates are usually detected by sequence-specific fluorogenic nucleic acid probes, generating corresponding fluorescence signals. 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 endpoint analysis, a "signal cluster" (also referred to as a population) in the data space can be assigned to a specific DNA or cDNA target sequence based on the fluorescence signal intensity, which enables an alternative type of data classification (Quan et al. 2018; Whale et al. 2016).

[0004] A relevant aspect of dPCR is the multiplexer, as different nucleic acid target sequences often occur in very low concentrations in samples, and it is highly important to detect as many target sequences as possible within a sample. The most common multiplexing methods in dPCR are based on DNA reporter molecules generating a fluorescence signal for each DNA target sequence in a spectral range that corresponds to the detection range of a detection channel. Thus, one DNA target sequence can be detected per device-side detection channel. However, the degree of multiplexing of a device is initially limited to the number of available detection channels. Currently available dPCR devices have between 2-6 different fluorescence detection channels, meaning a maximum of 6 target sequences can be analyzed on the device.A major problem here is that wavelengths can overlap when there are a large number of detection channels, and the resulting crosstalk results in lower specificity and sensitivity.

[0005] One method for increasing the degree of multiplexing uses combinatorial approaches, e.g., by combining Taqman probes and DropOff probes with specific reporter molecules as target sequences. In this case, the combinatorics of the signals generated by these probes also results in a higher degree of multiplexing ( (Madie et al. 2018; Stilla Technologies; Corne et al. 2021)). Since the individual mutations are sometimes only indirectly detected, these

[0006] REPLACEMENT SHEET (RULE 26) Methods are also very complex in their development and very demanding in their evaluation and certification or transfer to new target panels is correspondingly complex.

[0007] In addition, there are other methods for increasing the degree of multiplexing in digital PCR, which aim to generate additional populations in the data space. A US patent from Bio-Rad (US9921154B2) describes a multiplex digital PCR analysis of more target sequences than available optical channels, taking into account additional sample-specific aspects. However, the patent does not disclose any new assay features or methods for generating such target sequence populations. According to current knowledge, these methods can only be applied in cases where there is a significant multiple occupancy of droplets with different DNA target sequences or in cases where signals are generated by target sequence-specific reporter molecules such as Taqman probes or sequence-untargeted intercalating dyes, which in themselves do not represent an innovation compared to the state of the art.It has been described that different target sequence-specific probes can, for example, use different fluorophores as labels or can carry multiple fluorophore labels to generate multiple populations in the same detection channel. Since the signals of the probes described here are suppressed in the ground state by FRET quenching with low efficiency, the initial signal suppression is comparatively low, which is why effective modeling of the signal strength and thus separation of the populations in the data space is only possible to a limited extent. Since these labeled probes are also target sequence-specific, extensive optimization of their fluorescence properties is very inefficient, since new labeled probes must be synthesized each time, and this process must be started again when transferring to additional sequences.Likewise, the multiple modification of such target-sequence-specific probes with fluorophores is very complex and requires modification with additional quenchers to sufficiently suppress the signal in the ground state and avoid nonspecific signal populations in the data space. Such multiple modifications are very complex in terms of synthesis and usually require extensive optimization, which is why they are rarely used for target-sequence-specific reporter molecules, as this approach is very inefficient.

[0008] The most common variant of this intensity multiplexing is therefore the variation of the concentration of the different types of target sequence-specific reporter molecules (e.g., Taqman probes) while maintaining uniform and simple fluorescent labels for each detection channel (Fig. 1, right) (Pecoraro et al. 2016; Whale et al. 2016). This can result in additional populations in the data space of a graph. Due to the use of target sequence-specific fluorogenic reporter molecules, signal generation is directly dependent on the target sequence or the PCR system. Accordingly, this direct detection method is problematic due to the high optimization effort, as the strong rain (data points that lie between different populations) leads to blurred signal populations, which reduces precision (intensity multiplexing).The individual PCR components, and especially the fluorogenic reporters with identical fluorescent labels, must be well coordinated, e.g., in orthogonal concentration ratios, which is not always possible in practice. This method can also be supplemented by combining them with additional dyes that exhibit additional fluorescent labels. These dyes have their emission maximum between different fluorescence detection channels and can thus be detected equally in multiple fluorescence channels. This creates corresponding populations between the other clusters in the data space, which, however, becomes increasingly abstract as the data space expands due to additional detection channels. This makes the development of corresponding multiplex detection methods, and especially their approval, very complex.Another variation of intensity multiplexing uses even more complex concentration variations to stagger populations in the data space and thus make them less susceptible to rain (Hughesman et al. 2016).

[0009] A detection method for nucleic acids that, in contrast, does not use target sequence-specific reporter molecules and is based on a separation of nucleic acid detection by target sequence-specific mediator probes and signal generation by target sequence-unspecific universal reporters is the Mediator Probe PCR, which was patented by the University of Freiburg in 2012 (W02013079307A1, Faltin et al. 2012). This method is designed for colorimetric multiplexing with standardizable reporter molecules. In this method, one signal is recorded per detection channel, which, regardless of its signal strength, can be assigned to only one activated universal reporter in a homogeneous reaction.It has already been observed that under real-time PCR conditions, different fluorophores can lead to different signal intensities and signal curves, and that by maximizing the signal generation of the target sequence using nonspecific fluorogenic universal reporter molecules, the performance characteristics of the corresponding multiplex real-time PCRs can be optimized (Lehnert et al. 2018; Kipf et al. 2022). However, modeling the signal intensities in real-time PCR has no practical added value, as the signal curves in the same detection channel are indistinguishable using this approach. In colorimetric digital multiplex Mediator Probe PCR, it has also been shown that maximizing the fluorescence signal generation leads to better separation of the populations in the data space compared to the negative controls (Schlenker et al. 2021 b).

[0010] During the same period, Seegene Inc. developed a technology that also relies on the separation of DNA sequence detection and signal generation using two detection molecules in real-time PCR. An unlabeled PTO probe binds to a DNA target sequence, is cleaved, and releases a fragment, which then forms an extended duplex with a fluorescently labeled target sequence-nonspecific detection molecule (CTO molecule). This process is used to influence signal generation across different lengths of these detection molecules. This enables, for example, the differentiation of multiple target sequences in the same detection channel by reading the signal at defined, predefined temperatures.Another patent also uses readout at different temperatures in real-time PCR in one batch within the same detection channel and can therefore be interpreted as a further development of the aforementioned Seegene technology (PCT / CN2018 / 084794). Here, too, an extended reporter molecule is melted after detection. In contrast to the previous patent, a reporter molecule can be used to differentiate between different target sequences. Since temperature control during readout cannot currently be implemented as standard in commercially available dPCR devices, a direct transfer to dPCR is not possible. Hahn-Schickard had already worked on increasing the degree of multiplexing in PCR by generating virtual fluorescence channels via so-called photobleaching (WO2016087637A1) (Schuler et al. 2016).To combine this approach with the advantages of separating signal generation and detection in digital Mediator Probe PCR, a step was added following a digital Mediator Probe PCR using various universal reporter types with different fluorophore labels. In this step, these were bleached using LED light (bleaching) and read out in the same fluorescence detection channel. Depending on the fluorophore selected, a decrease in signal intensities can be observed due to bleaching, which allows the populations in the data space of a diagram to be separated and generates additional, so-called virtual channels (Schlenker et al. 2021a). However, since the bleaching technology requires additional devices and process steps that cannot currently be delivered with commercially available devices, implementation in existing dPCR platforms is not possible due to technical and regulatory hurdles.However, in this approach, the fluorophores selected as labels for the universal reporters did not yet result in different fluorescence signal intensities prior to the bleaching step, which would allow for consistent separation of the populations in the data space of a diagram when more than one target sequence was contained per reaction. The universal reporters used did not have the necessary configurations of fluorophores and quenchers that enable monochrome identification. Due to the high scatter of the fluorescence signal intensities of the reporter labels, a clear distinction between a single fluorescence signal population with a higher "rain" and a second signal population in the data space could not be achieved.In particular, this technology does not allow for obvious evaluation of whether a multiplex reaction involves one or two populations and thus does not provide the possibility of direct multiplexing in the same detection channel by target sequence-unspecific reporters (Schlenker et al. 2021a).

[0011] OBJECT OF THE INVENTION

[0012] Approaches to adjust the signal intensities of target sequence-independent reporter molecules (generated by, for example, different fluorophores and / or quenchers) in such a way that several distinguishable signal clusters are generated in one reaction in the same detection channel or in the corresponding area of ​​the multidimensional data space, thereby exploiting the advantages of indirect signal generation with regard to assay precision and robustness in order to circumvent the previously described problem of colorimetric multiplexing or target sequence-specific intensity multiplexing in a digital PCR, are currently lacking in the state of the art. So far, it has only been described to monochrome multiplex non-specific reporter molecules in a digital amplification by target sequence and thereby achieve higher multiplexing values ​​by introducing additional process steps (e.g., bleaching). This complicates the process.This complicates the evaluation, reduces precision, leads to additional optimization effort, 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-based detection channels.

[0013] SUMMARY OF THE INVENTION The present invention solves this problem by the method of the appended and independent claims.

[0014] Therefore, in one aspect, the invention relates to a method for the specific detection of at least two nucleic acid target sequences by at least two target sequence-independent reporter molecules in the same detection channel, comprising the steps: a. Providing at least a first and a second nucleic acid target sequence, b. Providing at least a first and a second mediator probe, each comprising an oligonucleotide, wherein the oligonucleotide of a first mediator probe comprises a mediator sequence and a probe sequence, wherein the mediator sequence has an affinity for a target sequence-independent reporter molecule of a first type and the probe sequence has an affinity for a first nucleic acid target sequence, wherein the oligonucleotide of a second mediator probe comprises a mediator sequence and a probe sequence,wherein the mediator sequence has an affinity for a target sequence-independent reporter molecule of a second type and the probe sequence has an affinity for a second nucleic acid target sequence, wherein the at least first and second mediator probes have no labels, or preferably no signal-generating labels, c. Providing at least two target sequence-independent reporter molecules of at least a first and a second type, each comprising at least one label with a respective measurable signal in the same detection channel, and a nucleic acid sequence, each of which has a specific affinity for at least one mediator sequence, wherein each type of the at least two target sequence-independent reporter molecules is characterized by the respective at least one label in such a way that it each has a signal intensity,which is preferably unique for the respective type of target sequence-independent reporter molecule, and is distinguishable from the signal intensities of the labels of all other target sequence-independent reporter molecule types and enables direct assignment to the respective nucleic acid target sequence, d. Carrying out a nucleic acid detection reaction, wherein at least one mediator sequence of at least one first mediator probe is released when its probe sequence binds to at least one first nucleic acid target sequence, wherein the at least one released mediator sequence binds to the at least one target sequence-independent reporter molecule of a first type, wherein a signal is generated by the at least one label which, through its signal intensity and / or emission spectrum, is characteristic of the first nucleic acid target sequence assigned to the respective target sequence-independent reporter,to which the at least one probe sequence of the at least one first mediator probe has bound, and optionally wherein at least one mediator sequence of at least one second mediator probe is released when its probe sequence binds to at least one second nucleic acid target sequence, wherein the at least one released mediator sequence binds to the at least one target sequence-independent reporter molecule of a second type, wherein the at least one label generates a signal which, through its signal intensity and / or emission spectrum, is characteristic of the second nucleic acid target sequence assigned to the respective target sequence-independent reporter, to which the at least one probe sequence of the at least one second mediator probe has bound, e. Detection of the signal(s) generated under step d., comprising the detection of the signal(s) in a detection channel and / or an analysis of the signal(s),the signal strength(s) and / or the emission spectrum(s) of the signal(s) and / or the cluster(s) of the signal(s) thus generated in a data space, preferably wherein the signals of different target sequence-unspecific reporter molecules can also be differentiated by their signal strengths in identical detection channels or the corresponding areas of a data space.

[0015] In embodiments, a first mediator probe thus comprises an oligonucleotide, which itself comprises a mediator sequence, and a probe sequence. In embodiments, a second mediator probe further comprises an oligonucleotide, which itself comprises a mediator sequence, and a probe sequence. Preferably, the mediator sequence of the first mediator probe differs from the mediator sequence of the second mediator probe. Preferably, the probe sequence of the first mediator probe differs from the probe sequence of the second mediator probe.

[0016] In other words, in embodiments of the method according to the invention, the mediator sequence of the first mediator probe differs from the mediator sequence of the second mediator probe, and the probe sequence of the first mediator probe differs from the probe sequence of the second mediator probe. Therefore, the probe sequence of the second mediator probe preferably also has an affinity for a "second" nucleic acid target sequence, which differs from the "first" nucleic acid target sequence (e.g., in its nucleic acid sequence), to which the probe sequence of the first mediator probe in turn has an affinity. The same applies to optionally present and optionally used additional (third, fourth, etc.) mediator probes.

[0017] In embodiments, step c comprises providing at least two target sequence-independent reporter molecules of at least a first and a second type, each comprising at least one label with a respective measurable signal in the same detection channel and / or a respective signal intensity maximum in the same detection channel, and a nucleic acid sequence, each of which has a specific affinity for at least one mediator sequence. According to the invention, a "first" nucleic acid target sequence and a "second" nucleic acid target sequence preferably differ in their nucleic acid sequence and / or their epigenetic modifications. The same also applies to optionally present and optionally detectable further (third, fourth, etc.) nucleic acid target sequences.

[0018] In preferred embodiments 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 another oligonucleotide during the nucleic acid detection reaction and thereby generate a signal. Different reporter molecule types are each modified with different labels (e.g., fluorophores and / or quenchers and / or fluorophores and quenchers in different numbers) and have different DNA sequences, which indirectly enable assignment to a target sequence. In identical detection channels, different target sequence-independent reporter molecule types generate signals of different signal intensities due to their different labels.This allows the signals of different reporter molecule types, each with different labels, to be differentiated and assigned to target sequences based on the different signal intensities, even in the same detection channel or the corresponding spanned data space (spanned by the axes in a multidimensional representation or by the different detection channels in a purely mathematical comparison / analysis and / or an algorithmic analysis). According to the invention, the signals of at least two different reporter complexes can preferably be detected in the same detection channel or the corresponding area of ​​a data space. The fact that these reporter molecules are independent of the target sequence enables, for the first time, the development of reporter molecules with particularly well-distinguishable signal intensities within the same detection channel.Because these labels are target sequence-independent, they can also possess suitable label configurations that efficiently enable such signal modeling. For example, their signals can be suppressed in the initial state by contact quenching, which results in greater differences in signal generation in the activated state, or they can possess multiple labels with fluorophores and / or quenchers. Users can thus increase the number of detectable target sequences in a reaction beyond the number of detection channels without having to purchase additional equipment, expand equipment, or implement further complex process steps. This represents enormous added value and ensures high cost efficiency. Existing laboratory workflows can be maintained, and already certified equipment can continue to be used without the need for re-implementation and, if necessary, recertification.Furthermore, this enables efficient optimization of these target-sequence-independent reporter molecules, as they can subsequently be used for additional target sequences and do not have to be developed and optimized from scratch, depending on the target sequence. Thus, the invention achieves simplified and efficient process optimization for the standardized detection of various target sequences and a reduction in the required labor and cost.

[0019] The method according to the invention enables precise, efficient (direct) monochrome multiplexing using target sequence-unspecific reporter molecules. This enables efficient target sequence-independent optimization of the reporter molecules, thus improving the separation of signal populations in the same detection channel or the corresponding area of ​​the data space. This enables precise detection of additional nucleic acid target sequences beyond the number of detection channels of the device. This is possible because the use of target sequence-independent reporter molecules enables symmetrical multiple labeling with fluorophores and quenchers and / or labeling with different fluorophore labels for the same detection channel in a suitable configuration and / or modeling of the fluorescence signal generation, e.g.Contact quenching is significantly simplified and improved compared to target sequence specific reporter molecules.

[0020] This is done using target-sequence-independent reporter molecules (e.g., universal reporters). These are labeled oligonucleotides that can each be (indirectly) assigned to a nucleic acid target sequence to be detected and that are activated by the binding of a released additional oligonucleotide during a nucleic acid detection reaction (e.g., PCR or digital PCR). To distinguish between the signals that represent / indicate the presence of different nucleic acid target sequences in the same detection channel or in the corresponding area of ​​the data space (the graphical representation of the detected signals), at least two target sequences to be detected are each assigned to two different types of target-sequence-unspecific reporter molecules, which generate a signal with different signal strengths in the same detection channel.

[0021] An advantage of the method according to the invention is the possibility of simultaneously detecting multiple target nucleic acid sequences in the same detection channel of a (PCR) device, e.g., the channel for detecting a red fluorescence signal. This simultaneous detection in a single detection channel is achieved by using different target sequence non-specific reporter molecules, each comprising labels that generate different signals with similar fluorescence emission spectra and that can be simultaneously detected in the same fluorescence detection channel of a device, e.g., a digital PCR device, while still being distinguishable from one another. This feature enables the invention to achieve monochrome multiplexing, namely the detection of multiple targets within a single (“monochrome”) fluorescence detection channel (with a specific wavelength range).

[0022] In some prior art digital PCR methods, a first or a second target sequence, each individually in a drop, is detected in emulsion droplets by using two labeled probes, each generating a homogeneous signal that can be distinguished from non-specific amplifications. In the method according to the invention, no labeled probes are used, which has the advantage that the labeled reporter molecules can be optimized to a greater extent than would be possible with the known prior art methods, since the reporter molecules according to the invention can subsequently be used for further assays. In addition, the reporter molecules according to the invention can preferably only adopt one configuration in which contact quenching occurs. This preferred property preferably enables or supports (direct) monochrome multiplexing in the form according to the invention.A further advantage of the method according to the invention is that the desired detection properties can be precisely adjusted by selecting the respective reporter molecules used, which are independent of the target sequence to be detected. The easy-to-produce and design link between reporter molecules with desired properties (e.g., fluorescence intensity and / or color) are the mediator probes, which comprise a probe and a mediator sequence. To individualize the target nucleic acid to be detected, only the two different sequence components of the mediator probe need to be provided. These can then be combined with the target sequence-nonspecific reporter molecule matching the mediator sequence. This independence allows for the desired specific contact quenching to be adjusted, enabling the detection of several distinguishable reporter molecule signals in the same detection channel.Furthermore, signal optimization can be performed once, independent of numerous target sequences. Furthermore, the method according to the invention is resource-efficient, since reporter molecule sets can be reused for any number of nucleic acid target sequences, thus making the initially time-consuming development of combinable reporter molecule signals very labor- and cost-efficient.

[0023] In addition, the method according to the invention enables direct detection of a nucleic acid target sequence since it can be directly assigned to a data population (signal population in the evaluation / analysis) and does not require combinatorics.

[0024] One possibility to generate different signal intensities by different labels on reporter molecules arises, for example, from the physical molecular properties of the labels, which are amplified by contact quenching after activation of the target sequence-unspecific reporter molecule:

[0025] In embodiments, the at least one label of a target sequence-independent reporter molecule is therefore at least one fluorophore and / or at least one quencher.

[0026] Another essential property of these target sequence non-specific reporter molecules for the efficient generation of precisely distinguishable signal populations is the selection of a labeling configuration that enables signals to be generated in such a way that their signal intensities are sufficiently robust, stable, and thus precise enough to remain distinguishable during the readout process without further processing steps (such as bleaching). It has been shown that this is possible, for example, by consistently using configurations corresponding to the basic structure of a universal reporter or corresponding modifications. Target sequence non-specific reporter molecules can be appropriately modeled, allowing different signal intensities to be precisely adjusted in the detection channel (Lehnert et al. 2018).

[0027] 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. In embodiments, the at least one label of a target sequence-independent reporter molecule comprises at least two fluorophores and / or two quenchers with the same or different emission spectrum and / or the same or different signal intensity.

[0028] In some embodiments, a reporter molecule of a first type differs from that of at least a second type by the number of labels or the number of fluorophores and / or quenchers. Thus, different types of target-sequence-independent reporter molecules can be distinguished based on the signal strength and / or intensity of the fluorescence released upon their activation. For this purpose, different types of target-sequence-independent reporter molecules can comprise a different number and / or different fluorophores (with different emission spectra / colors and / or intensities) and / or quenchers, or any combination thereof.

[0029] In preferred embodiments in which the label comprises at least one fluorophore, its fluorescence signal is suppressed until the reporter molecule is activated, preferably by contact quenching. Thus, preferably no signal is released by a non-activated reporter molecule. Activation preferably occurs through the binding of a mediator sequence to the corresponding target sequence-independent reporter molecule and / or through subsequent extension of a mediator sequence already bound to the target sequence-independent reporter molecule by a polymerase during a nucleic acid detection reaction.

[0030] In embodiments, therefore, the at least one label of a target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher, wherein contact quenching preferably takes place between the at least one fluorophore and the at least one quencher as long as no mediator sequence binds to the corresponding target sequence-independent reporter molecule, or as long as a bound mediator sequence has not been extended during the nucleic acid detection reaction.

[0031] This enables, for example, the detection of additional DNA target sequences in a suitable device within the context of digital DNA amplification and fluorescence-based detection, without the need for additional technical aids or the need to vary the concentrations of reporter molecules with fluorescent labels. This allows, for example, similar results to those achieved with digital Mediator Probe PCR (MP-PCR) in combination with photobleaching to be generated without the need for a bleaching process (e.g., via an LED or thermal). This significantly reduces and simplifies the experimental effort and makes it feasible even in existing devices.The method according to the invention thus represents enormous added value compared to digital Mediator Probe PCR (MP-PCR) or other state-of-the-art methods, as it is directly compatible with existing platforms and can significantly simplify potential diagnostic approval of a specific detection method. The method according to the invention offers additional new degrees of freedom in assay development, while the synthesis processes for corresponding target sequence-specific probes do not necessarily become more complicated. Furthermore, the sensitivity of an assay according to the invention can be significantly increased, as each reaction can detect additional nucleic acid sequences.A further advantage is that a suitable set of target sequence non-specific reporter molecules always generates populations in similar areas of the data space, which allows conclusions to be drawn about the respective activated reporter independent of the target sequences within the detection reaction and thus the same set can be used with different nucleic acid target sequence panels without complex optimization.

[0032] In embodiments of the method according to the invention, the analysis of the signal, the signal strength and / or the emission spectrum of the signal under point e. comprises the representation and / or analysis of the detected signal depending on the signal strength and / or the detection channel and / or the emission spectrum in a data space spanned by the evaluated detection channels.

[0033] In the context of the present invention, a data space is preferably generated by a plot, a diagram or a graph of the different detection channels or by a purely mathematical and / or algorithm- and / or computer-based evaluation, comparison or contrast of the different detection channels, whereby the respective fluorescence signals of the target sequence-unspecific reporters used generate data points in this 1 - n dimensional data space (depending on the number of detection channels required for the evaluation), which can be grouped into clusters (groups, accumulations).

[0034] In the context of the present invention, a data space is preferably the space which, in a plot, diagram, or graph, is spanned by an X-axis and a Y-axis (two-dimensional plot), and optionally, in a three-dimensional graph, additionally by a Z-axis, or, in a correspondingly higher-dimensional space, by a further axis, and in which the data to be displayed (the detected signals) are represented. In embodiments in which detection channels used in the method are represented on the axes of a graph, for example, the signals detected in the red detection channel on the X-axis and the signals detected in the green detection channel on the Y-axis, the data space can also be referred to as "spanned by the detection channels."Preferably, it is possible to unambiguously assign the detected signals displayed in the data space, which can preferably be represented as distinct signal populations, to a target sequence. This is preferably also possible if several signals specific to different target sequences were detected in the same detection channel.

[0035] In embodiments, the reporter molecules used simultaneously in a PCR reaction differ from one another either by different types (e.g., colors, emission spectra, and / or intensities) of fluorophores and / or different types of quenchers and / or by the number of labels on the different reporter molecules. In embodiments of the monochrome multiplexing according to the invention, at least two target nucleic acids are indirectly detected in the same detection channel by reporter molecules with different or distinguishable signals.If more than two target nucleic acids are to be detected, in embodiments, the signals to be generated for the detection of the target nucleic acids can, if necessary, be divided between multiple detection channels (and the labels of the different reporter molecule types are selected accordingly), with preferably at least two target nucleic acids being detected indirectly in the same detection channel by reporter molecules with different or distinguishable signals. In embodiments, at least one first and one second nucleic acid target sequence, or at least one first, one second, and one third, or even a fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, or even more target sequences can be detected, or their presence can be detected.

[0036] Depending on the number of nucleic acid target sequences to be detected, at least one first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first or even more mediator probes are provided, each comprising an oligonucleotide, each comprising a mediator sequence and a probe sequence.

[0037] Accordingly, depending on the number of nucleic acid target sequences to be detected, target sequence-independent reporter molecules of a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first or even more type are provided and used in the method or kit according to the invention as described herein.

[0038] A person skilled in the art will understand how to combine types of target sequence independent reporter molecules and their labels according to the invention to achieve the detection of desired target sequences according to the invention.

[0039] In embodiments, the label of the at least one target sequence independent reporter molecule comprises at least two complementary opposite nucleobases, each with at least one label, or at least two opposite bases offset by one base position from a complementary base pairing, each with at least one label.

[0040] The present invention enables the simultaneous detection of multiple target nucleic acids (multiplexing), whereby 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 comprise labels that generate distinguishable signals despite the simultaneous detection of the signals of several different reporter molecules in the same detection channel and can be represented during analysis as distinct (non-overlapping) signal populations in the two- or three-dimensional data space. In embodiments, this enables the differentiation of the signals indicating the presence of different target nucleic acids despite detection in the same detection channel, as well as, in specific embodiments, also the differentiation from background noise.

[0041] In embodiments of the method according to the invention, in step d.-e., n different nucleic acid target sequences are indirectly detected by n different target sequence-independent reporter molecule types, wherein the detection of the signal generated in step d. takes place in k detection channels, where n > k and and wherein at least two different target sequence-independent reporter molecule types are detected in step e. in the same detection channel and / or are displayed in the same area of ​​a data space in the display under point e.

[0042] By applying the method according to the invention, it is possible in embodiments to perform data classification in higher-dimensional data space (>3 dimensions, with more than three detection channels). Using the method according to the invention, the number of distinguishable target sequences in a digital PCR can be at least doubled compared to the available detection channels, provided this is not inherently restricted by device-related technical limitations, such as possible crosstalk (mutual influence of fluorescence signals; erroneous association of a fluorescence signal with the signal of another fluorophore due to the erroneous detection of fluorescence from a neighboring detection channel) between different detection channels.

[0043] In embodiments, a signal from a label of a reporter molecule is suppressed, deleted, or quenched until a mediator sequence binds to the reporter molecule and is preferably extended by a polymerase in a nucleic acid amplification reaction. In embodiments, the polymerase achieves a spatial separation, e.g., of at least one fluorophore from at least one quencher, so that a signal is generated and can be detected.

[0044] In embodiments of the method according to the invention, the signal of the labeling of a target sequence-independent reporter molecule is generated by the 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.

[0045] In embodiments, the at least one target sequence independent reporter molecule is an oligonucleotide or an oligonucleotide complex.

[0046] In embodiments of the method according to the invention, the nucleic acid detection reaction under step d. comprises an amplification method for DNA and / or cDNA.

[0047] In embodiments of the method according to the invention, the nucleic acid detection reaction under step d. is a PCR, RT-PCR, RPA or LAMP, and wherein, as part of the DNA amplification, a mediator sequence of a mediator probe bound to a target nucleic acid is released by an enzymatic activity of a biomolecule, which then binds to a target sequence-independent reporter molecule, so that a signal is generated.

[0048] In embodiments, the detection under step e. is carried out as part of a digital amplification and / or signal generation.

[0049] In embodiments of the method according to the invention, the target sequence-independent reporter molecules are universal reporters and / or modular reporter complexes and the (respectively) at least one released mediator sequence is a component of a mediator probe PCR or mediator displacement LAMP.

[0050] In embodiments, the nucleic acid target sequence is derived from the conversion of another biomolecule into DNA sequence information. In some embodiments, the presence and / or amount of an RNA sequence (an embodiment of a biomolecule) in a sample can be determined by transcribing the RNA sequence into cDNA using a reverse transcription reaction. In embodiments, this cDNA can be amplified within the scope of the method according to the invention and indirectly detected using a target sequence-independent reporter molecule.

[0051] In embodiments of the method according to the invention, the at least one marking comprises a marking selected from an electrochemically active and / or a magnetic marking.

[0052] In embodiments of the method according to the invention, the signal is read out at different temperatures.

[0053] In some preferred embodiments, a mediator probe comprises an oligonucleotide and a sequence-specific probe segment that binds to the target sequence and is protected at the 3' end. This protection at the 3' end can be a blocking group (protecting group), e.g., a chemical blocking or protecting group, which in some embodiments comprises a chain of three carbon atoms. Protection of the mediator probe at the 3' end preferably prevents (non-specific) extension of the sequence strand by a polymerase during an amplification reaction. According to the invention, in embodiments, the mediator probe can comprise any protecting or blocking group suitable for preventing (non-specific) extension of the mediator probe sequence strand by a polymerase during an amplification reaction.In some embodiments, the mediator probe is protected against (non-specific) polymerase extension by means other than a blocking group (protecting group) at the 3' end.

[0054] In other embodiments, a mediator probe does not comprise a blocking group (protecting group) at the 3' end and is not protected against (non-specific) polymerase extension.

[0055] In embodiments, a Mediator probe protected at the 3' end may comprise a "C3 spacer." Such a C3 spacer may be a chemical block group, which in some embodiments comprises a chain of three carbon atoms. This "C3 spacer" thus preferably prevents (nonspecific) polymerase extension of the Mediator probe sequence strand. Typical block groups (protecting groups) that are suitable depending on the embodiment are known to those skilled in the art. Based on the present disclosure of the invention, the skilled person also knows how to select suitable block groups (protecting groups) as routine adaptations of the invention described herein.

[0056] Preferably, the method according to the invention thus enables direct endpoint detection of n targets in k detection channels, where n > k and n 2. In contrast to the prior art, signals are not generated by fluorogenic probes, but by target sequence-independent reporter molecules, which can also be described as population-specific reporters, which preferably differ clearly in the intensity range. This ensures greater flexibility in the experimental design process and enables the use of reporter molecules that utilize contact quenching for initial signal suppression. Furthermore, it enables controlled signal generation, resulting in more distinguishable and robust fluorescence signals that are particularly suitable for endpoint detection. Preferred embodiments of the present invention are based on universal reporter structures and are used in combination with mediator probes for target sequence detection.One of the basic principles of the method according to the invention is preferably the detection of n target nucleic acid sequences by n target-specific mediator probe types that activate n reporter molecule types, whereby each reporter molecule type is labeled with a unique fluorophore with defined optical properties (Figure 1). In an example of a duplex reaction, two fluorophores would be selected that emit fluorescence in the same wavelength range but differ in their intensity in the detection range. During dPCR amplification, the bound label-free mediator probes are cleaved by the polymerase and release a mediator sequence. This mediator activates a reporter molecule type, which then generates a fluorescent signal.The sum of all droplets or compartments with a signal of a specific reporter molecule type forms a population in a specific region of the data space, corresponding to the unique fluorescent label. Reporter molecule types labeled with fluorophores with higher quantum yields generate positive populations with higher fluorescence intensities (Figure 1, bottom: high intensities are shown as larger circles), and those with lower quantum yields in the detection region generate positive populations with lower fluorescence intensities (Figure 1, top: low intensities are shown as smaller circles). Depending on their position in the data space, the formed populations can be assigned to a specific target DNA sequence to be detected in the sample.In the presence of two targets in a compartment, an additive effect of fluorescence intensities is assumed, which would lead to the formation of a third population with higher signal intensity. In further embodiments, a similar signaling effect could be observed, for example, by using two different quencher types or two similar fluorophore labels on a reporter molecule in a duplex reporter molecule dPCR reaction. In an example of a four-plex reporter molecule dPCR assay performed in two detection channels, the presence of multiple targets in a compartment leads to the formation of orthogonal populations. This means that by using four different reporter molecule types, theoretically up to sixteen different populations could be detected and differentiated (e.g., Figure 5).Without being bound by theory, it can be assumed that in digital assays, the distribution of a target nucleic acid follows Poisson statistics. Thus, the generation of double and multiple positive populations could depend on the concentrations of the target nucleic acid, as well as on potentially occurring competing effects caused by the detection of gene loci that share the same primer pairs. Compared to state-of-the-art higher-order multiplexing methods, the present method has the advantage that the concentrations of the target-independent reporter molecules can remain constant across assays. This allows greater freedom in assay design, and the fluorescence signal populations can be optimized with high efficiency. A further advantage of combining mediator probes with target-independent reporter molecules comes into play in SNP detection.During the elongation process, preferentially bound Mediator probes are highly specifically cleaved by the polymerase, with the Mediator portion being cleaved from the probe portion. The cleavage site is preferably located between the first and second nucleotides of the probe portion of the Mediator probe. Only if the cleavage was performed correctly can the released Mediator fully bind to the Mediator-binding site on the specific target-sequence-independent reporter molecule type to initiate signal generation. If proper cleavage has not occurred, the existing 3' overhang will preferentially hinder the elongation process on the target-sequence-independent reporter molecule, and no signal is generated. Therefore, in the case of SNP detection, the first nucleotide can preferentially be positioned at the 5' end of the target-specific probe portion of a Mediator probe above the SNP position to activate the specific target-sequence-independent reporter molecule type.

[0057] In a further aspect, the invention relates to a kit comprising: at least one target sequence-independent reporter molecule of a first type comprising at least one label, optionally at least one target sequence-independent reporter molecule of a second type comprising at least one label, which preferably emits a signal in the same detection channel or the corresponding region of a data space as the at least one target sequence-independent reporter molecule of a first type, optionally at least one first mediator probe, whose mediator sequence has an affinity for the at least one target sequence-independent reporter molecule of a first type, and whose oligonucleotide sequence has an affinity for a first nucleic acid target sequence, optionally at least one second mediator probe, whose mediator sequence has an affinity for the at least one target sequence-independent reporter molecule of a second type,and whose oligonucleotide sequence has an affinity to a second nucleic acid target sequence, optionally at least one buffer, optionally a polymerase, optionally a reverse transcriptase, optionally at least one PCR primer.

[0058] In one embodiment, the kit according to the invention is suitable or configured for carrying out the method according to the invention.

[0059] In embodiments, a kit according to the invention can comprise more than two different types of target sequence-independent reporter molecules and optionally also more than one first and one second mediator probe, the mediator sequence of which has an affinity to the respective target sequence-independent reporter molecules of a first or second or further type, and the oligonucleotide sequence of which accordingly each has an affinity to a first or a second or further nucleic acid target sequences, so that more than two different nucleic acid target sequences can be specifically detected.

[0060] The embodiments described for one aspect of the invention may also be embodiments of any of the other aspects of the present invention. Accordingly, embodiments described for the method according to the invention may also be embodiments of the kit according to the invention. Furthermore, each embodiment described herein may also include features of any other embodiment of the invention. The various aspects of the invention are united by, benefit from, are based on, and / or are linked to the common and surprising discovery of the unexpected advantageous effects of the present method, namely the optimized simultaneous PCR detection of multiple target sequences by target sequence-independent reporter molecules.

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] The term "target sequence-independent reporter molecule" describes a molecule or complex consisting of at least one DNA oligonucleotide for signal generation during PCR in the presence of DNA target sequences. A target sequence-independent reporter molecule preferably comprises at least one label and at least one quencher.

[0063] In embodiments, a target sequence-independent reporter molecule comprises one or more Mediator binding sites. Thus, a target sequence-independent reporter molecule can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or even 50 Mediator binding sites. In preferred embodiments, a target sequence-independent reporter molecule comprises between 1 and 10 Mediator binding sites. An (activated) mediator or mediator sequence bound to a mediator binding site can, when extended by a (PCR) polymerase, lead to the activation or degradation, digestion, cleavage or release of one or more labels, e.g. quenchers and / or fluorophores, from one or more modifications located preferably upstream (towards the 5' end) on the reporter molecule.

[0064] In the context of the present invention, mediator nucleic acid sequences can be referred to both as mediator probes and with the English term "mediator probe" or "mediator probe." This always refers to nucleic acid probes that transmit a signal between a target nucleic acid homologous to the mediator probe and a target sequence-independent reporter molecule. During a nucleic acid amplification reaction, e.g., PCR or digital PCR reaction, a polymerase migrating forward on the target nucleic acid can cleave or digest a mediator probe bound to the target nucleic acid. A mediator sequence, also simply referred to as "mediator," is then released, which can bind to a mediator binding site of a target sequence-independent reporter molecule. If a polymerase binds to the reporter molecule during the PCR and begins to extend the bound mediator sequence, a label is preferably activated, e.g.,preferably a quencher separated from a fluorophore so that a signal is generated and / or a signal change is effected. In other words, herein a "mediator" or "mediator sequence" refers to a nucleic acid oligonucleotide that can be extended by the polymerase along the target sequence-independent reporter molecule. A "mediator probe" describes a nucleic acid oligonucleotide, preferably a DNA oligonucleotide, that establishes the connection between the target sequence and the target sequence-independent reporter molecule by binding to it in the presence of a DNA target sequence during PCR, being cleaved by the exonuclease activity of the polymerase, and releasing a mediator sequence, which then binds to an associated target sequence-independent reporter molecule for which it has an affinity.

[0065] In the context of the present invention, affinity to a nucleic acid target sequence describes a physical attraction that two homologous nucleic acid sequences have for each other, which can lead to them binding to each other or hybridizing with each other. For affinity or successful hybridization of two homologous nucleic acid sequences, in preferred embodiments, there is complete homology between two nucleic acid sequences of a certain length, such as between a probe sequence and a target nucleic acid sequence. In other embodiments, the homologous sequences can contain at least one mismatch to each other.

[0066] In the context of the present invention, a data space is preferably generated by a plot, a diagram or a graph of the different detection channels or by a purely mathematical, algorithm- and / or computer-based evaluation, comparison or contrast of the different detection channels, whereby the respective fluorescence signals 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 the evaluation), which can be grouped into clusters (groups, accumulations).Thus, in embodiments, a "data space" can be the space which is generated or "spanned" in a plot, diagram, or graph, preferably by an X-axis and a Y-axis (two-dimensional plot), and optionally - in a three-dimensional graph - additionally by a Z-axis, or in higher dimensions additionally by another axis, and in which the data to be displayed is represented. In embodiments in which the signals detected in the respective detection channels are displayed on the axes of a graph or plot - for example, the signals of the red detection channel on the X-axis, the signals of the green detection channel on the Y-axis, and the signals of the blue detection channel on the Z-axis of the plot - the data space can also be referred to as being "spanned by the detection channels."In further embodiments, a data space is generated by a purely algorithm- and / or computer-based evaluation, comparison and / or comparison of the different detection channels and / or the signals detected therein and is preferably "spanned" by the analyzed detection channels.

[0067] By applying the method according to the invention, it is possible in embodiments to perform data classification in higher-dimensional data space (>3 dimensions, with more than three detection channels). In embodiments, the method according to the invention can at least double the number of distinguishable target sequences in a digital PCR compared to the available detection channels, provided this is not inherently restricted by device-related technical limitations, such as possible crosstalk between different detection channels.

[0068] In the context of the present invention, a "label" may comprise one or more fluorophores and / or one or more quenchers. Accordingly, in the context of the invention, reporter molecules may carry or comprise one or more fluorophores and / or quenchers. The proximity of a fluorophore to the quenchers prevents the detection of its fluorescence. In embodiments, the reporter-quencher proximity is interrupted, e.g., by release of one or more fluorophores and / or quenchers, which in embodiments occurs by partial degradation of the reporter molecule, e.g., at the 5' end, by hydrolysis by the 5' to 3' exonuclease activity of the PCR polymerase used for the amplification reaction. This enables unquenched emission of fluorescence, which can be detected after excitation with a laser.In preferred embodiments, a target sequence-independent reporter molecule comprises at least one fluorophore and at least one quencher (these are therefore preferably present in pairs), whereby the quencher preferably suppresses the fluorophore signal until a mediator sequence binds to the reporter molecule and is preferably extended by a polymerase in the context of a nucleic acid amplification reaction. In embodiments, the polymerase achieves a spatial separation, e.g., of at least one fluorophore from at least one quencher, so that a signal is generated and can be detected. In embodiments in which multiple quencher-fluorophore pairs are present within a target sequence-independent reporter molecule, the pairs can also be arranged at different locations relative to one another.A target sequence independent reporter molecule may comprise one or more labels, for example, 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 may be present. In some embodiments, a target sequence independent reporter molecule comprises 1, 2, 3, or up to 5 labels. In other embodiments, a target sequence independent reporter molecule comprises 1, 2, 3, 4, 5, or even more than 5 labels.

[0069] For the purposes of the invention, a "C3 spacer" is preferably a chemical block group (protecting group), which preferably comprises a chain of three carbon atoms. This block group (protecting group) preferably serves to prevent (non-specific) polymerase extension of the strand. Typical and—depending on the embodiments—suitable C3 spacers / block groups (protecting groups) are known to the person skilled in the art, and the person skilled in the art knows, based on the present disclosure of the invention, how to select suitable C3 spacers / block groups (protecting groups) as routine adaptations of the invention described herein.

[0070] According to the invention, a signal-generating label is preferably a fluorophore or other dye capable of generating a detectable signal. In some embodiments, a signal-generating label is an electroactive or magnetic label. In the context of some embodiments, the terms "signal-generating label" and "label" are equivalent or interchangeable.

[0071] In the context of the invention, a biomolecule preferably comprises nucleic acids.

[0072] The term "nucleic acid" refers to nucleic acid molecules, including, without limitation, DNA, ssDNA, dsDNA, RNA, mRNA, tRNA, lncRNA, ncRNA, microRNA, siRNA, rRNA, sgRNA, piRNA, rmRNA, snRNA, snoRNA, scaRNA, gRNA, or viral RNA. Nucleic acid sequences refer herein to a sequential arrangement of nucleotides, where nucleotides are represented by their nucleobases in guanine (G), adenine (A), cytosine (C), and thymine (T) in DNA and uracil (U) in RNA. A nucleic acid sequence may also refer herein to the sequence of consecutive letters or nucleobases (consisting of G, A, C, and T or U) that represent the actual sequence of consecutive nucleic acids in a DNA or RNA strand. This nucleic acid sequence can be identified and characterized biochemically and bioinformatically using DNA or RNA sequencing or by complementary nucleic acid probes (e.g.In embodiments herein, mediator probes) can be specifically detected, e.g., in the context of a PCR, real-time PCR, or a detection reaction of a digital PCR. Sequence analysis can also comprise the comparison of the resulting nucleic acid sequence or a detection signal specific thereto with one or more reference nucleic acid sequences and / or with the detection signals of housekeeping genes. The term nucleotide can be abbreviated to "nt." The term base pair (two nucleobases bound to each other via hydrogen bonds) can be abbreviated to "bp."

[0073] A “target sequence” (also referred to herein as “target”) in the context of the invention describes any nucleic acid sequence of interest that is to be detected by the method according to the invention. A target sequence can preferably be a DNA, cDNA, cfDNA, or RNA sequence. A target sequence can represent part or all of a target DNA nucleic acid sequence. A mediator probe preferably comprises a sequence that is fully or partially complementary to the nucleic acid sequence of the target sequence or a part thereof. In some embodiments, this sequence of the mediator probe is 100%, 99%, 95%, 90%, or 80% complementary to the target sequence. In some embodiments, a mediator probe can tolerate one or more mismatches to the target sequence and still bind to it. In other embodiments, the mediator probe only binds to a target sequence if it matches the target sequence 100%.

[0074] The term "nucleic acid amplification reaction" refers to any method that involves an enzymatic reaction that enables the amplification of nucleic acids. A preferred embodiment of the invention relates to a polymerase chain reaction (PCR). The polymerase chain reaction ("PCR") is the gold standard method for rapidly producing millions to billions of copies (complete copies or partial copies) of a given DNA sample, thereby enabling the amplification of a very small DNA sample to a sufficiently large amount. PCR amplifies a specific region of a DNA strand (the target DNA sequence), depending on where the primers used bind to initiate the amplification reaction. Almost all PCR applications use a heat-stable DNA polymerase enzyme, such as Taq polymerase.

[0075] In digital PCR (dPCR), the reaction mix and the nucleic acids it contains (e.g., ctDNA or DNA) are divided into thousands of reaction chambers, separating the target sequences from one another and facilitating their amplification and detection, particularly of rare target sequences. A common method is the microfluidic dropletization of the reaction mix containing the nucleic acid to be detected in oil or the distribution into fixed microcavities, whereby each formed unit forms a closed reaction chamber for amplification and detection, for example, during PCR. If a nucleic acid target sequence is present in a droplet, it is amplified by PCR (or other amplification methods). The individual amplification products are usually detected using sequence-specific fluorogenic nucleic acid probes, generating corresponding fluorescence signals.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 endpoint analysis, a "signal cluster" (also called a population) in the data space can be assigned to a specific DNA or cDNA target sequence based on the fluorescence signal intensity, enabling an alternative type of data classification (Quan et al. 2018; Whale et al. 2016).

[0076] "Multiplexing" generally means detecting multiple target sequences simultaneously in a single reaction without the need to further divide the reaction mixture for the detection of multiple target sequences. In "multiplex PCR," several individual PCR reactions for different DNA sequences or genes are combined under identical conditions into a single reaction mixture. By allowing multiple sequences to be analyzed simultaneously, information can be obtained from a single PCR reaction that would otherwise require many times more reagents and more time to perform. Thus, "multiplexing" reduces the cost and time required to perform a PCR because fewer reagents are used per experiment, experiments can be performed more quickly, and results can be analyzed more quickly.Since splitting a reaction mixture allows a target sequence to be detected to be divided into a single detection reaction in which it would not be detected, multiplex detection methods are more sensitive and therefore very promising detection methods, especially in oncology diagnoses. Since pipetting errors can be minimized in one reaction compared to several, the use of multiplex PCR can also improve precision compared to single assays.

[0077] Quantitative PCR (“qPCR”) or “real-time PCR” (“real-time PCR”) represents a specific form of PCR and is a standard method for detecting and quantifying a specific target sequence or for quantifying gene expression levels in a sample in real time. In qPCR, fluorescently labeled probes or nucleic acids (e.g., mediator probes) are hybridized in the PCR reaction and, in some embodiments, cleaved or digested by the PCR polymerase during primer extension as soon as they bind to a complementary sequence (e.g., a target sequence). In some embodiments, the presence and amplification of target sequences is monitored in real time after or during each PCR cycle. Real-time PCR makes it possible to monitor the progress of an ongoing amplification reaction as it occurs (i.e., in real time). Data is therefore collected throughout the entire PCR reaction and not at the endpoint as with conventional PCR.Measuring reaction kinetics in the early stages of PCR offers significant advantages over conventional PCR detection. In real-time PCR embodiments, reactions are characterized by the time point during the cycle at which amplification of a target is first detected, rather than by the amount of target accumulated after a fixed number of cycles, as in classical PCR. The higher the starting copy number of the nucleic acid target, the more likely a significant increase in fluorescence is to be observed. Real-time PCR enables analysis using optical signals to detect a specific PCR product (the target sequence), using specific fluorochromes or fluorophores. An increase in DNA product during PCR therefore leads to an increase in the fluorescence intensity measured at each cycle.Using different colored labels, fluorescent probes can be used in multiplex assays to monitor multiple target sequences. While real-time qPCR depends on determining the relative amount of the target nucleic acid in each amplification cycle, digital PCR, on the other hand, allows for the determination of the absolute amount of the target nucleic acid based on Poisson statistics, which is applied following endpoint PCR amplification to calculate the target nucleic acid amount. The steps prior to amplification are usually comparable or similar between digital PCR and qPCR. However, in qPCR, all nucleic acid molecules are preferably pooled and then amplified and analyzed, whereas in digital PCR, the nucleic acid molecules are preferably distributed as best as possible across individual partitions (e.g.Emulsion drops, wells, or gel beads), allowing the PCR to run as a single reaction in each partition (in the case of emulsion drops, this reaction is often referred to as droplet PCR or digital droplet PCR) and allowing each partition to be analyzed separately. The random distribution of nucleic acid molecules into individual partitions in digital PCR follows the Poisson distribution. When analyzing the digital PCR, Poisson statistics are then applied to determine the average number of nucleic acid molecules per partition (none, one, or more). The statistical Poisson analysis of the number of positive and negative reactions provides precise absolute quantification of the target sequence.

[0078] Recombinase polymerase amplification (“RPA”) is a method for amplifying (replicating) DNA and is a variant of isothermal DNA amplification. RPA is typically performed using a recombinase (single-strand binding protein) and a strand-shifting DNA polymerase, with the recombinase increasing primer binding. When a strand-shifting polymerase is used, it is possible to perform the reaction at 37 to 42°C or even at room temperature. By adding a reverse transcriptase enzyme to an RPA reaction, reverse transcription can be performed. In various variants of RPA, the resulting DNA can optionally be additionally quantified (similar to qPCR) and / or multiple DNA sequences can be amplified in parallel in a multiplex process.

[0079] The term "loop-mediated isothermal amplification" or "LAMP" refers to a method for amplifying DNA, with LAMP also being a variant of isothermal DNA amplification. An isothermal amplification reaction usually takes place at a constant temperature. This property distinguishes LAMP from PCR, in which the reaction takes place with a series of alternating temperature steps or cycles. In LAMP, a strand-shifting DNA polymerase is usually used. In a LAMP reaction, for example, four to six primers can bind to six to eight DNA sequences, which requires special primer design. In a LAMP reaction, reverse transcription can be carried out by adding a reverse transcriptase. In addition, the DNA produced in a LAMP reaction can be quantified.It is also possible to detect several DNA sequences in parallel in a multiplex LAMP reaction.

[0080] In the context of the invention, a "signal change" describes a fluorescence signal change. This signal change is preferably a significant, differentiable, and / or characteristic change in the fluorescence signal, which is clearly separated or distinguished from potential baseline or background signals or background or background noise. The person skilled in the art is aware that under some experimental conditions, nonspecific fluorescence baseline signals or background or background noise can occur due to fluorophores during fluorescence detection. Therefore, a signal change in the context of the invention preferably describes a significant, differentiable, and / or characteristic change in the fluorescence signal, and not a fluorescence baseline or background signal or background or background noise. In preferred embodiments, this signal change can mean an increase in fluorescence intensity, in other words, an increase in the fluorescence signal.In some embodiments, a signal change is the decrease in the fluorescence signal. The increase in a fluorescence signal is preferably due to the fact that an amplification reaction increases the number of target sequence amplicons and thus the activation of associated signal complexes. Accordingly, the number of resulting cleavages, digestions, and / or separations of the respective signal oligos from their binding site on the associated base strands increases, whereby at least one fluorophore is released and / or separated from its quencher (i.e., the distance between quencher and fluorophore increases such that the fluorescence signal is no longer quenched by the quencher). An increase (increase in the number) of released and / or non-quenched fluorophores thus leads to an increase in the fluorescence signal, which is specific and indicative for a specific target sequence.The more target sequences are present and bound by mediator probes in a PCR reaction, the more the fluorescence signal increases. Preferably, the fluorescence signal is proportional or approximately proportional to the amount of the corresponding target sequence for which the fluorophore signal (e.g., its color) is specific / characteristic. Since, in a digital or droplet PCR, preferably only one target sequence is present per reaction chamber (e.g., partition, emulsion droplet), the signal increases increasingly with the number of target sequence amplifications per reaction chamber until a signal plateau is reached, which is determined by the maximum fluorescence intensity of the reporter molecules used for the respective detection. In preferred embodiments, there is ideally a uniform distribution of max. 1 target sequence per reaction chamber (e.g.,Partition, emulsion droplets) at the beginning of amplification, so that with a similar amplification efficiency and similar maximum fluorescence intensity of the detection molecules in all reaction chambers (containing a target sequence), the specific signal for the detection of an identical target sequence in different reaction chambers is generated with comparable height / strength / intensity during readout by digital or "droplet" PCR, which is preferably indicative of the presence and / or number of target sequence amplicons present in each reaction chamber. In embodiments, the intensity / strength of a respective label, preferably specific for a target sequence, as well as the maximum achievable signal strength / intensity can depend on the number of labels per signal oligo and signal complex and / or the type of label (e.g., type of fluorophore and / or quencher).

[0081] “Fluorophore” (or fluorochrome, similar to a chromophore) is a fluorescent chemical compound that can re-emit light when excited by light. Fluorophores for use as labels in the construction of labeled probes of the invention include, but are not limited to, rhodamine and derivatives such as Texas Red, fluorescein and derivatives such as 5-bromomethylfluorescein, Lucifer Yellow, IAEDANS, 7-Me2N-coumarin-4-acetate, 7-OH-4-CH3-coumarin-3-acetate, monobromobimane, pyrene trisulfonates such as Cascade Blue and monobromotrimethyl-ammoniobimane, 7-NH2-4CH3-25-coumarin-3-acetate (AMCA), FAM, TET, CAL Fluor Gold 540, JOE, VIC, Quasar 570, CAL Fluor Orange 560, Cy3, NED, Oyster 556, TMR, CAL Fluor Red 590, HEX, ROX, LC Red 610, CAL Fluor Red 610, Texas Red, LC Red 610, CAL Fluor Red 610, LC Red 640, CAL Fluor Red 635, Cy5, LC Red 670, Quasar 670, Oyster 645, LC Red 705, Cy5.5, BODIPY FL, Rhodamine Green, Oregon Green 30 488, Oregon Green 514, Cal Gold, BODIPY R6Gj, Yakima Yellow, Cal Orange, BODIPY TMR-X, JOE, HEX, Quasar-570 / Cy3, TAMRA, Rhodamine Red-X, Redmond Red, BODIPY 581 / 591 , Cy3.5, Cal Red / Texas Red, BODIPY TR-X , BODIPY 630 / 665-X, Quasar-670 / Cy5, Pulsar-650, Dy490, Atto-488, Atto532, Atto-Rho-6G, Dy590, Atto-Rho101 , Cy5, Dy-636, Atto-647N, Cy5.5, Dy682, Atto-680, BMN-488, BMN-505, BMN-536, BMN-562,.

[0082] Quenching refers to any process that reduces the fluorescence intensity of a given substance. Quenching is the basis for Förster resonance energy transfer (FRET) assays, static or contact quenching assays, or a combination of both. FRET is a dynamic quenching mechanism because the energy transfer occurs while the donor is in an excited state. Contact quenching requires close spatial proximity in the form of physical contact between the donor and quencher. A quencher is a molecule that quenches the fluorescence emitted by the fluorophore when it is excited by the light source of a PCR cycler or detection device.Quenchers for use as labels in the construction of labeled signal oligos and / or base strands of the invention include, without claiming to be exhaustive, DDQ-I, Iowa Black, Iowa Black FQ, QSY-9, BHQ-1, QSY-7, BHQ-2, DDQ-II, 22 Eclipse, Iowa Black RQ, QSY-21, BHQ-3 Dabcyl, QSY-35, BHQ-0, ElleQuencher, BMN-Q1, BMN-Q2, BMN-Q60, BMN-Q-535, BMN-Q590, BMN-Q620, BMN-Q650. The person skilled in the art knows suitable reporter-quencher pairs and knows which to select for a particular application.

[0083] EXAMPLES OF IMPLEMENTATION

[0084] The invention is further described by the following examples. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention, which are provided to illustrate the invention described herein.

[0085] Methods

[0086] In the following embodiments, PCR amplifications were carried out in the form of digital PCR. Digital PCR refers to a PCR amplification reaction in which individual nucleic acid molecules are preferably distributed across individual partitions (e.g., emulsion droplets, wells, or gel beads), whereby the PCR proceeds as a single reaction in each partition (in the case of emulsion droplets, this reaction is often also referred to as droplet PCR or digital droplet PCR) and enables separate analysis of each partition. This analysis is preferably carried out by fluorescence analysis (e.g., by laser excitation of a fluorophore and detection of its emitted fluorescence) of each individual partition. If a target sequence is present in a partition and is detected by the method according to the invention, this is preferably indicated by a specific fluorescence signal.

[0087] The Naica system from Stilla Technologies was used for the examples presented. In the first step, the droplets were generated and thermally processed using the Geode. The dPCR protocol used was 5 minutes at 95°C, followed by 45 cycles of 15 cycles at 95°C and 60 cycles at 58°C. The Prism 3 reader and the CrystalMiner software developed for it (version 3.1.6.3) were used to evaluate the droplets in examples 1-8, and the Prism 6 reader was used in example 9.

[0088] For working examples 1-8, single-strength PerfeCTa® Multiplex qPCR ToughMix® with 50 nM Alexa Fluor 488 was used as the background fluorophore. In duplex and quadruple-plex reactions, 0.5 pM forward and 0.25 pM reverse primers, 1.2 pM mediator probes, and 0.6 pM target sequence independent reporter were used for the respective target sequences. The concentration of the target sequence independent reporter was reduced to 0.4 pM for the six-plex reaction. For working example 9, single-strength naica® multiplex PCR MIX 10X was used, and 1.2 pM mediator probes and 0.4 pM target sequence independent reporter, as well as a total of 2 pM forward and 1 pM reverse primers, were used for the respective target sequences.

[0089] In embodiment 10, the procedure can be analogous to embodiment 9, whereby mediator probes for the ZURs used in embodiment 9 would be adapted to further target sequences.

[0090] To prevent nonspecific extension of relevant oligonucleotide sequences, chemical protecting groups are preferably used to modify the 3' end of an oligonucleotide sequence. These are, for example, chains comprising three carbon atoms (C3 spacers), which are chemically attached and block extension of an oligonucleotide sequence by a polymerase during a PCR reaction.

[0091] Sequences:

[0092] Table 1: Primers

[0093] Table 2: Mediator probes. In the sequences, the probe regions are shown in capital letters; the bases relevant for the point mutation are marked in bold. A C3 spacer is preferably a chemical block group comprising a chain of three carbon atoms to prevent nonspecific polymerase elongation of the strand.

[0094] Table 3: Target sequence independent reporter molecules (The fluorophore modifications are located internally at the first 3' base of the stem-loop structure (nt 22 (7; 6) or nt 20 (8); represented as 6, 7 or 8); quenchers were attached to the first base of the stem-loop structure on the 5' side (nt 1 )). A C3 spacer is preferably a chemical block group comprising a chain of three carbon atoms to prevent nonspecific polymerase extension of the strand.

[0095]

[0096] Table 4: Target sequences (primer hybridization regions are underlined;

[0097] Point mutations / SNPs are shown in bold)

[0098] Example 1

[0099] In the present example, a signal is generated when a mediator probe binds to a nucleic acid target sequence and is digested or cleaved by a polymerase during a PCR reaction, which amplifies a nucleic acid sequence. This releases the mediator, also called the mediator sequence, and allows it to bind to a target-sequence-independent reporter molecule. If the bound mediator is recognized by a polymerase during the PCR reaction as a primer or starting oligonucleotide for amplification, bound by the polymerase, and extended, a quencher or fluorophore located downstream (downstream of the amplification direction) from the mediator binding site is separated or displaced, resulting in a signal generated by a fluorophore that was previously quenched by contact quenching.This signal is characteristic of the nucleic acid target sequence to which the mediator probe (mediator probe) has previously bound. The method according to the invention enables (direct) monochrome multiplexing in digital PCR by using at least two target sequence-independent reporter molecules (see, for example, “target sequence-independent reporter” in Figure 1), each with different fluorophore modifications (see, for example, Figure 1, shown therein as a circle or triangle) with an emission maximum in one (e.g., in the red) detection channel in a configuration that enables signal identification. In Figure 1, different fluorescence signal intensities are represented by circles of different sizes.By using target sequence-independent reporter molecules, contact quenching can be achieved more efficiently than with state-of-the-art target sequence-specific Taqman probes, thereby amplifying or more easily modeling the differences in signal strength of different fluorophores in the same detection channel.

[0100] For (direct) multiplexing, i.e. the simultaneous detection of different target sequences in a sample to be analyzed and / or in a reaction, the current state of the art usually requires multiple optical channels, additional process steps, or complex concentration adjustment or modification of the reporter molecules. In contrast, the method according to the invention makes it possible to detect different target sequences in a sample in the same detection channel without being limited by the points listed above. The use of target sequence-independent reporter molecules also enables the reuse of optimized designs for different target sequences and makes their application more efficient.different intensity combinations and / or different fluorophore colors of the respective markers, preferably different extended mediator sequences and thus to encode and differentiate different detected target sequences.

[0101] In embodiments, multiple target sequences can be detected simultaneously using different fluorescence intensities and / or fluorescence colors by using different target sequence-independent reporter molecules. These target sequence-independent reporter molecules each have different mediator binding sites with fluorescence and / or quencher labels of different signal intensities and / or colors / emission spectra. However, preference is given to using target sequence-independent reporter molecules, each of which can generate at least two reporter molecules that can be measured / detected in the same detection channel.

[0102] By generating signals with different signal intensities, these can be distinguished in a fluorescence channel. These embodiments offer significant advantages, particularly in digital PCR, for distinguishing different signal clusters and represent an improvement over the state of the art.

[0103] Example 2

[0104] An example in which two target-sequence-independent reporter molecules, each with different fluorophore and quencher numbers and an emission maximum in the red detection channel, are used is another example of (direct) monochrome multiplexing according to the present invention. In this example, different fluorescence signal intensities are generated by different numbers of fluorophores during digital PCR, thus enabling signal identification. By using target-sequence-independent reporter molecules, symmetric multiple modification of fluorophores and quenchers can be used more efficiently, for example, in contrast to target-sequence-specific Taqman probes, thereby amplifying the differences in signal intensities of different fluorophores in the same detection channel.

[0105] Example 3

[0106] In a sample analysis, (direct) monochrome multiplexing was performed using two target-independent reporter molecules with different fluorophore labels in the red detection channel. The two different target-independent reporter molecules were labeled with either Cy5 or Atto-647N. The analysis included an amplification reaction within a digital PCR (mediator probe PCR) on the Stilla Naica system, with the obtained results being able to be graphically displayed in multiple dimensions of a data space.

[0107] Figure 2 shows a graphical evaluation and representation of the digital PCR results in the form of a 1-D (one-dimensional) plot.

[0108] Figure 3 shows a graphical evaluation and representation of the digital PCR results in the form of a 2D (two-dimensional) plot. The specific signals of the reporter complexes / reporter molecules in the red channel ("red", Y-axis) were compared with the 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 complexes / reporter molecules are clearly distinguishable from each other as distinct signal populations in the two-dimensional data space, and from the background noise (in the blue detection channel).

[0109] Figure 4 shows a graphical evaluation and representation of the digital PCR results in the form of a 3D (three-dimensional) plot. The specific signals of the reporter complexes / reporter molecules in the red channel (“red”, Y-axis) were compared with both the background signal in the blue detection channel (“blue”, X-axis) and the signals of a negative control (sample without target gene) in the green channel (“green”, Z-axis) in a three-dimensional data space. In this embodiment, too, the two different signals of the reporter complexes / reporter molecules are clearly distinguishable from each other as distinct signal populations in the three-dimensional data space, and from the background noise and a negative control signal—not detected here but potentially present in other analyses.

[0110] Example 4

[0111] In the present experiment, a digital PCR reaction using mediator probes is performed by employing four different types of target sequence-independent reporter molecules (ZUR1-ZUR4) with different fluorescent labels. A (direct) monochrome multiplexing is performed in two detection channels (e.g., red and green channels). When graphically displaying the signals detected during the digital PCR reaction, which are generated by the different types of reporter molecules, these can be represented as signal populations in the data space (the space generated or spanned by an x-axis and a y-axis and in which the data is graphically displayed).

[0112] Figure 5 shows a theoretical scheme of the representation of an analysis of signals from an example with four different reporter molecules (ZUR1-ZUR4), where different florescence signal intensities are represented by circles of different sizes and positions in the data space. The data space in Figure 5 is spanned by the x-axis, on which the intensity of detected signals in the red detection channel (ZUR4 and ZUR2) is shown, and the y-axis, on which the detected signal intensity in the green detection channel (ZUR3 and ZUR1) is shown. The signal populations in the data space can be clearly assigned to one of the four types of activated target sequence-independent reporter molecules and could therefore, in contrast to target sequence-specific reporter molecules (e.g., Taqman probes), be efficiently used to detect different target sequence panels (target sequence compilations or combinations).

[0113] Example 5

[0114] In a further embodiment of the invention, samples in the red, green, and blue detection channels could be analyzed in parallel during a (direct) monochrome mediator probe (mediator probe) multiplex PCR. For this purpose, the 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 to a duplex reaction (a reaction mixture comprising two different reporter molecule types, each specific for one of the two DNA target sequences), so that either both target sequences could be detected simultaneously, or only one or none of them could be detected in the respective detection channel.

[0115] Figure 6 shows the results of this exemplary embodiment for the use of two different reporter complexes / reporter molecules in the red detection channel in a (direct) monochrome multiplexing analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, with the leftmost data space containing the signals characteristic of target sequence 1 and target sequence 2 (X-axis). The middle data space shows the signals of the reactions with the single addition of a target sequence (individual reactions in the duplex reaction mixture), which are specific for the individual target sequence 1 (X-axis) or target sequence 2 (X-axis). The rightmost data space shows the measured signal for the negative control (NTC; X-axis). The Y-axis shows the detected signal intensity in the red detection channel.For signal generation, two types of target sequence-independent reporter molecules (ZUR05 and ZUR06) were used, either labeled with Cy5 / BHQ-2 or BHQ-2 / Atto-647N.

[0116] The different signals of the reporter complexes / reporter molecules are easily differentiated from each other as distinct signal populations in the two-dimensional data space, so that specific detection of both target sequences was possible.

[0117] Figure 7 shows the results of this exemplary embodiment for the use of two different reporter complexes / reporter molecules in the green detection channel in a (direct) monochrome multiplexing analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, with the detected signals specific for target sequence 1 and target sequence 2 (X-axis) being shown in the leftmost data space. The middle data space shows the two individual detections after adding only one DNA target sequence each for the individual detection of target sequence 1 (X-axis) and target sequence 2 (X-axis). The negative control (NTC; X-axis) is shown in the rightmost data space. The signal intensity in the green detection channel is shown on the Y-axis.For signal generation, two types of target sequence-independent reporter molecules (ZUR02 and ZUR04) were used, either labeled with Dy530 and BMN-Q1 or with BMN536 and BHQ-1.

[0118] The different signals of the reporter complexes / reporter molecules are easily differentiated from each other as distinct signal populations in the two-dimensional data space, so that specific detection of both target sequences was possible.

[0119] Figure 8 shows the results of this exemplary embodiment for the use of two different reporter complexes / reporter molecules in the blue detection channel in a (direct) monochrome multiplexing analysis in the Stilla Naica system. The signals of a duplex reaction (double reaction) are shown, with the leftmost data space containing the signals specific for target sequence 1 and target sequence 2 (X-axis). The middle data space shows the two individual detections after adding only one DNA target sequence each, indicating the presence of the individual target sequence 1 (X-axis) or target sequence 2 (X-axis). The rightmost data space shows the signal of the negative control (NTC; X-axis). The Y-axis shows the signal intensity in the blue detection channel.For signal generation, two types of target sequence-independent reporter molecules (ZUR07 and ZUR08) were used, either labeled with FAM / BHQ-1 or with Atto488 / BMN-Q1 .

[0120] The different signals of the reporter complexes / reporter molecules are easily differentiated from each other as distinct signal populations in the two-dimensional data space, so that specific detection of both target sequences was possible.

[0121] Example 6

[0122] In a further embodiment of the invention, samples in the green and blue detection channels could be simultaneously analyzed for the presence of four target sequences during a (direct) monochrome mediator probe (mediator probe) multiplex PCR. For this purpose, the samples were amplified and analyzed in a suitable digital PCR system, such as the Stilla Naica system. In this "4-plex mediator probe PCR," four target-sequence-independent reporter molecules and four target-sequence-specific mediator probes were provided 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).The reporter molecules for KRAS target gene variants (different BRAF or KRAS single nucleotide polymorphisms (SNP) or wild-type (WT) variants) generated a signal in the green detection channel (BMN536 and Dy-530 fluorophore) upon activation, the reporter molecules for BRAF target gene variants generated a signal in the blue detection channel (FAM and Atto 488 fluorophore) upon activation.

[0123] Figure 9 shows the result of this SNP analysis. The signals of target sequences I and II detected individually in a partition are shown in the upper left quadrant of the data space. The signals of target sequences III and IV detected individually in a partition are shown in the lower right quadrant of the data space. The signals of one KRAS and one BRAF target sequence (i.e., I+III, II+III, I+IV, and II+IV) detected simultaneously in a partition are shown in the upper right quadrant of the data space. The Y-axis shows the signal intensity in the green detection channel (BMN536 and Dy-530 fluorophore), and the X-axis shows the signal intensity in the blue detection channel (FAM and Atto 488 fluorophore).

[0124] As can be seen in Figure 9, several signal clusters can be detected per detection channel, which can be assigned to activated, target sequence-independent reporter molecules and thus to the corresponding target sequences.

[0125] Example 7

[0126] In another embodiment of the invention, samples could be analyzed simultaneously for the presence of six target sequences in the green, red, and blue detection channels during a (direct) monochrome mediator probe (mediator probe) multiplex PCR. For this purpose, the samples were amplified and analyzed in a suitable digital PCR system, such as the Stilla Naica system. In this 6-Plex Mediator Probe PCR, six target sequence-independent reporter molecules and six target sequence-specific mediator probes were provided to detect 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). Upon activation, the reporter molecules for BRAF target gene variants BRAF V600E and WT generated a signal in the blue detection channel (FAM and FAM, respectively).Atto 488 fluorophore), the reporter molecules for KRAS WT and G12A target gene variants generated a signal in the green detection channel upon activation (DY-530 and BMN-536 fluorophore, respectively), and the reporter molecules for KRAS G12D and G12V target gene variants generated a signal in the red detection channel upon activation (Cy5 and Atto-647N fluorophore, respectively).

[0127] Figure 10 shows the result of this analysis, with the signals of target sequences I and II detected individually in a partition shown on the x-axis of the data space. The signals of target sequences V and VI detected individually in a partition are shown on the y-axis of the data space. The signals of target sequences III and IV detected individually in a partition are shown on the z-axis of the data space. The data space also shows the signals of the respective KRAS and BRAF target sequences detected simultaneously in a partition. The y-axis shows the signal intensity in the red detection channel, the x-axis the signal intensity in the blue detection channel, and the z-axis the signal intensity in the green detection channel.

[0128] As can be seen in Figure 10, several signal clusters can be detected per detection channel, which can be assigned to activated, target sequence-independent reporter molecules and thus to the corresponding target sequences.

[0129] Example 8

[0130] By applying the described method, it is also possible to perform data classification in higher-dimensional data spaces (>3 dimensions with more than three detection channels). The described method can theoretically at least double the number of distinguishable target sequences in a digital PCR compared to the available detection channels, provided this is not inherently restricted by device-related technical limitations such as crosstalk between different detection channels:

[0131] The person skilled in the art is familiar with appropriate methods for classifying higher-dimensional data structures.

[0132] Example 9

[0133] In a further embodiment, the multiplexing capacities per detection channel can be increased beyond doubling using the described method. Thus, multiplexing capacities per detection channel can be at least tripled or even further increased. This is made possible by using at least three reporter molecules in a detection channel, which generate at least three distinguishable populations in the data space in the respective detection channel, which are specifically assigned to each reporter. This allows up to three target sequences to be detected in a detection channel, tripling the device-side detection channel capacities, provided they are not inherently restricted by device-side technical limitations such as crosstalk between different detection channels. Figure 12 shows the results of such a tripling of the detection channels in the Stilla Prism-6.The presence of three target sequences was analyzed simultaneously in the infrared detection channel of the Stilla Prism-6. For this purpose, the samples were amplified and analyzed in a suitable digital PCR system, such as the Stilla Naica system. In this "3-Plex Mediator Probe PCR," three target sequence-specific mediator probes were provided to detect the three target sequences I - KRAS G12D (KRAS single point mutation G12D), II KRAS WT (KRAS wild-type genome sequence), and III KRAS G12V (KRAS single point mutation G12V) using the three target sequence nonspecific reporter molecules with the fluorophore-quencher combinations Cy5.5 + BHQ2 (I KRAS G12D), Dy636 + BHQ2 (II KRAS WT), and Atto680 + BHQ2 (III KRAS G12V).A particular advantage of this method over the state-of-the-art is that signal generation using target sequence-nonspecific reporter molecules makes signal generation very robust. Populations are generated in the data space at comparable intervals across multiple independent experiments, thus simplifying data analysis. This increases the benefit of the signal optimization required for such population separation.

[0134] Example 10

[0135] In a further embodiment, due to the separation of the detection of one or more DNA target sequences by cleavage of one or more different mediator probes combined with the 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, a 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 are adapted to the different DNA target sequences by exchanging the mediator probes and / or primers.In this way, the efficiency of optimizing fluorescence signals from target-independent reporters is significantly higher, as these only need to be optimized once per detection device (e.g., a digital PCR thermocycler) compared to the state of the art. This allows for the generation of many different, highly differentiated fluorescence signal populations in the data space more efficiently than before, as this optimization only needs to be performed once and can then be transferred to additional DNA target sequence panels. Corresponding optimization processes are thus more efficient and generally known to those skilled in the art and described in the literature (Lehnert et al. 2018).This means that assays that utilize direct monochrome multiplexing can achieve better performance in terms of sensitivity, precision, or specificity with less effort thanks to the optimized signals than state-of-the-art assays, whose signals must be re-optimized for each new target sequence. Compared to the state-of-the-art, this has the particular advantage that a signal can always be unambiguously assigned to the same reporter based on its position in the data space alone, and correlation of this position in the data space with further information, such as temperature or the position of other signal populations in the data space, is not necessary to determine activation of the respective target sequence-nonspecific reporter.

[0136] FIGURES

[0137] The invention is further described by the following figures. These are not intended to limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention provided to illustrate the invention described herein.

[0138] Figure 1: Direct monochrome multiplexing in digital PCR using two target-independent reporter molecules (“ZUR”) of a first (A) and a second (B) type, each with a different fluorophore label (shown as a circle or triangle) with an emission maximum in the red detection channel in a configuration that allows signal identification. Different fluorescence signal intensities, which are characteristic of a particular nucleic acid target sequence, are represented by circles of different sizes. By using target-independent reporter molecules, contact quenching (of the target, a fluorophore, by the target, a quencher) can be achieved more efficiently than with target-sequence-specific Taqman probes, thereby amplifying the differences in signal intensities between different fluorophores in the same detection channel.The 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 and activated by a specific mediator sequence (“Mediator 1” under (A) or “Mediator 2” under (B)), which was previously cleaved and / or released from the corresponding mediator probe (“Mediator Probe 1” under (A) or “Mediator Probe 2” under (B)).

[0139] Figure 2: The graph represents the results of Example 3 using a one-dimensional plot (1D plot). In Example 2, two nucleic acid target sequences were detected by a (direct) monochrome ("Mediator Probe") duplex PCR (digital PCR in the Stilla Naica system) using corresponding specific target sequence-independent reporter molecule types of a first and a second type. The two different reporter molecule types included either the red fluorophore Cy5 or the red fluorophore Atto 647N as a label, which were characteristic of the respective nucleic acid target sequence and were detected and analyzed in the red detection channel of a Stilla Naica system (digital PCR system).

[0140] Figure 3: The graph represents the results of Example 3 using a two-dimensional (2D) plot. In Example 2, two nucleic acid target sequences were detected by a (direct) monochrome ("Mediator Probe") duplex PCR (digital PCR in the Stilla Naica system) using corresponding specific target sequence-independent reporter molecule types of a first and a second type. The two different reporter molecule types included either the red fluorophore Cy5 or the red fluorophore Atto 647N as a label, which were characteristic of the respective nucleic acid target sequence and were detected in the red detection channel of a Stilla Naica system (digital PCR system). This plot compares the specific signals of the reporter complexes / reporter molecules in the red channel ("Red", Y-axis) with the background signal (background noise) in the blue detection channel ("Blue", X-axis) in a 2D data space.Y-axis unit: relative fluorescence units (RFU).

[0141] Figure 4: The graph represents the results of Example 3 using a three-dimensional (3D) plot. In Example 2, two nucleic acid target sequences were detected by a (direct) monochrome ("Mediator Probe") duplex PCR (digital PCR in the Stilla Naica system) using corresponding specific target sequence-independent reporter molecule types of a first and a second type. The two different reporter molecule types included either the red fluorophore Cy5 or the red fluorophore Atto 647N as a label, which were characteristic of the respective nucleic acid target sequence and were detected in the red detection channel of a Stilla Naica system (digital PCR system).This plot compares the specific signals of the reporter complexes / reporter molecules in the red channel (Y-axis) with the background signal (background noise) in the blue detection channel (Blue, X-axis) and the signals of a negative control (containing no target sequence) in the green detection channel (Green, Z-axis) in a 3D data space. Axis units: relative fluorescence units (RFU).

[0142] Figure 5: This shows a schematic of the experimental design and the reporter molecules used in Example 4. In Example 4, a digital PCR reaction using four different types of target sequence-independent reporter molecules (ZUR1-ZUR4) with different fluorescent labels is performed, performing direct monochrome multiplexing in two detection channels (e.g., red channel (x-axis) and green channel (y-axis)). The four different types of target sequence-independent reporter molecules (ZUR1-ZUR4) are each bound and activated by a specific mediator sequence (mediator type 1-4), with the generated and detected fluorescence signal being specific for a respective nucleic acid target sequence.

[0143] Figure 6: The results of Example 5 are shown. Two different target sequence-independent reporter molecule types were used in the red detection channel in a (direct) monochrome multiplexing PCR analysis in the Stilla Naica system. The signals from a duplex reaction (double reaction) are shown, with the leftmost data space containing the signals indicating the presence of target sequence 1 and target sequence 2 (X-axis). The middle data space shows the signals from the individual detections after adding only one DNA target sequence each, which are specific for target sequence 1 (X-axis) and target sequence 2 (X-axis). The rightmost data space shows the signal of the negative control (NTC; X-axis). The Y-axis shows the signal intensity in the red detection channel (“Red”).Two types of target-independent reporter molecules (ZUR05 and ZUR06) were used for signal generation, labeled either with Cy5 / BHQ-2 or BHQ-2 / Atto-647N. Y-axis unit: relative fluorescence units (RFU).

[0144] Figure 7: The results of this embodiment 5 for the use of two different reporter complexes / reporter molecules in the green detection channel in a (direct) monochrome multiplexing analysis in the Stilla Naica system are shown. The signals of a duplex reaction (double reaction) are shown, with the signals specific for target sequence 1 and target sequence 2 (X-axis) being shown in the leftmost data space. The middle data space shows the two individual detections after adding only one DNA target sequence each for the detection of the individual target sequence 1 (X-axis) or target sequence 2 (X-axis). The rightmost data space shows the signal of the negative control (NTC; X-axis). The Y-axis shows the signal intensity in the green detection channel.Two types of target-independent reporter molecules (ZUR02 and ZUR04) were used for signal generation, labeled either with Dy530 / BMN-Q1 or with BMN536 / BHQ-1. Y-axis unit: relative fluorescence units (RFU).

[0145] Figure 8: The results of this embodiment 5 for the use of two different reporter complexes / reporter molecules in the blue detection channel in a (direct) monochrome multiplexing analysis in the Stilla Naica system are shown. The signals of a duplex reaction (double reaction) are shown, with the leftmost data space containing the signals specific for target sequence 1 and target sequence 2 (X-axis). The middle data space shows the individual detections after adding only one DNA target sequence, which indicate the presence of the individual target sequence 1 (X-axis) or target sequence 2 (X-axis). The rightmost data space shows the signal of the negative control (NTC; X-axis). The Y-axis shows the signal intensity in the blue detection channel.Two types of target-independent reporter molecules (ZUR07 and ZUR08) were used for signal generation, labeled either with FAM / BHQ-1 or with Atto488 / BMN-Q1. Y-axis unit: relative fluorescence units (RFU).

[0146] Figure 9: The result of Example 6 is shown, in which samples in the green and blue detection channels were simultaneously analyzed for the presence of four target sequences using a (direct) monochrome mediator probe (mediator probe) multiplex PCR. For this purpose, the samples were amplified and analyzed in a corresponding digital PCR system (Stilla Naica System). In this "4-Plex 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). The reporter molecules for KRAS target gene variants generated a signal in the green detection channel (Y-axis) upon activation, while the reporter molecules for BRAF target gene variants generated a signal in the blue detection channel (X-axis) upon activation.In the illustrated 2D plot (2D data space), the signals of target sequences I and II detected individually in a partition are shown in the upper left quadrant of the data space. The signals of target sequences III and IV detected individually in a partition are shown in the lower right quadrant of the 2D data space. The signals of one KRAS and one BRAF target sequence (i.e., I+Ill, II+Ill, I+IV, and II+IV) detected simultaneously in a partition are shown in the upper right quadrant of the 2D data space. The Y-axis shows the signal intensity in the green detection channel, and the X-axis shows the signal intensity in the blue detection channel. The axes are plotted in relative fluorescence units (RFU).

[0147] Figure 10: This figure shows a three-dimensional (3D) plot of the results of Example 6. Using a (direct) monochrome mediator probe (mediator probe) multiplex PCR, samples were simultaneously analyzed in the green, red, and blue detection channels for the presence of six target sequences. For this purpose, the 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, label: FAM / BHQ-1 ), II - BRAF WT (BRAF wild-type gene; ZUR08, label: Atto 488 / BMN-W1 ), III - KRAS WT (KRAS wild-type gene; ZUR02, label: DY-530 / BMN-Q1 ), IV - KRAS G12A (KRAS gene mutation G12A; ZUR04, label: Cy5 / BHQ-1 ), V - KRAS G12D (KRAS gene mutation G12D; ZUR05, label: Cy5 / BHQ-2), VI - KRAS G12V (KRAS gene mutation G12V, ZUR06, labeling: Atto 647N / BHQ-2). The reporter molecules for BRAF target gene variants BRAF V600E and WT generated a signal in the blue detection channel upon activation (FAM and Atto 488 fluorophore, respectively), while the reporter molecules for KRAS WT and G12A target gene variants generated a signal in the green detection channel upon activation (DY-530 and DY-538, respectively).BMN-536 fluorophore) and the reporter molecules for KRAS G12D and G12V target gene variants generated a signal in the red detection channel (Cy5 and Atto-647N fluorophore, respectively) upon activation. In the 3D plot (3D data space), the signals of target sequences I and II detected individually in a partition are shown on the x-axis. The signals of target sequences V and VI detected individually in a partition are shown on the y-axis of the 3D data space. The signals of target sequences III and IV detected individually in a partition are shown on the z-axis of the 3D data space. The 3D data space also shows the signals of the respective KRAS and BRAF target sequences detected simultaneously in a partition. The Y-axis represents the signal intensity in the red detection channel, the X-axis the signal intensity in the blue detection channel, and the Z-axis the signal intensity in the green detection channel. The axes are expressed in relative fluorescence units (RFU).

[0148] Figure 11: One possibility for generating different signal intensities by different labels on reporter molecules arises in embodiments, for example, from the physical molecular properties of the labels, which are amplified by contact quenching after activation of the target sequence-non-specific reporter molecule. In both graphs, the fixed dimensions of a label ("marker") and the measured signal are shown on the x-axis, e.g. wavelength, potential, frequency, etc. The variable dimensions of the measured signal, in this case the signal strength, are shown on the y-axis. The points indicate the intersection point of the respective signal intensities 1 and 2 of the fluorophore 1 or 2 and the respective measurement wavelength.

[0149] Figure 12: Representation of the results from application example 9, where three different DNA target sequences were detected in one detection channel by three different target sequence-unspecific reporter molecules, each with three different fluorescent labels, in a 3-plex mediator probe PCR with three different mediator probes in the infrared channel of the Prism6. In this "3-Plex Mediator Probe PCR," the three target sequences I - KRAS G12D (KRAS single point mutation G12D), II KRAS WT (KRAS wild-type genome sequence), and III KRAS G12V (KRAS single point mutation G12V) were detected using the three target sequence nonspecific reporter molecules with the fluorophore-quencher combinations Cy5.5 + BHQ2 (I KRAS G12D), Dy636 + BHQ2 (II KRAS WT), and Atto680 + BHQ2 (III KRAS G12V). Each of these target sequence nonspecific reporter molecules thus generates a specific population in the data space, which can be assigned to one of the three DNA target sequences.

[0150] LITERATURVERZEICHNIS

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Claims

PATENT CLAIMS 1. Method for detecting at least two nucleic acid target sequences by at least two target sequence-independent reporter molecules in the same detection channel, comprising the steps of: a. providing at least one first and one second nucleic acid target sequence, b.Providing at least one first and one second mediator probe, each comprising an oligonucleotide, wherein the oligonucleotide of the first mediator probe comprises a mediator sequence and a probe sequence, the mediator sequence having an affinity for a first-type target sequence-independent reporter molecule and the probe sequence having an affinity for a first nucleic acid target sequence, wherein the oligonucleotide of the second mediator probe comprises a mediator sequence and a probe sequence, the mediator sequence having an affinity for a second-type target sequence and the probe sequence having an affinity for a second nucleic acid target sequence, wherein the at least first and second mediator probes do not have signal-generating labels, c.Providing at least two target sequence-independent reporter molecules of at least one first and one second type, each comprising at least one label with a measurable signal in the same detection channel, and a nucleic acid sequence, each possessing a specific affinity for at least one mediator sequence, wherein each type of at least two target sequence-independent reporter molecules is characterized by its at least one label in such a way that it possesses a signal intensity that is distinguishable from the signal intensities of the labels of all other target sequence-independent reporter molecule types and allows a direct assignment to the respective nucleic acid target sequence, i.e.Performing a nucleic acid detection reaction, wherein at least one mediator sequence of at least one first mediator probe is released when its probe sequence binds to at least one first nucleic acid target sequence, wherein the at least one released mediator sequence binds to at least one target sequence-independent reporter molecule of a first type, wherein the at least one label generates a signal which, by its signal intensity and / or emission spectrum, is characteristic of the first nucleic acid target sequence assigned to the respective target sequence-independent reporter to which the at least one probe sequence of the at least one first mediator probe has bound. e. Detection of the signal generated in step d., comprising detection of the signal in a detection channel and / or analysis of the signal, signal strength and / or emission spectrum of the signal.

2. Method according to claim 1, wherein the at least one label of a target sequence independent reporter molecule is at least one fluorophore and / or at least one quencher.

3. Method according to one of the preceding claims, wherein the analysis of the signal, the signal strength and / or the emission spectrum of the signal under point e. comprises the representation and / or analysis of the detected signal depending on the signal strength and / or the detection channel and / or the emission spectrum in a data space spanned by the evaluated detection channels.

4. Method according to one of the preceding claims, wherein different target sequence-independent reporter molecule types differ by the signal intensity and / or the emission spectrum of their at least one label.

5. Method according to any of the preceding claims, wherein the at least one label of a target sequence independent reporter molecule comprises at least two fluorophores and / or two quenchers with the same or different emission spectrum and / or the same or different signal intensity.

6. Method according to one of the preceding claims, wherein the at least one label of a target sequence independent reporter molecule comprises at least one fluorophore and at least one quencher, wherein contact quenching preferably takes place between the at least one fluorophore and the at least one quencher as long as no mediator sequence binds to the corresponding target sequence independent reporter molecule, or as long as a bound mediator sequence has not been extended during the nucleic acid detection reaction.

7. Method according to one of the preceding claims, wherein the labeling of the at least one target sequence independent reporter molecule comprises at least two complementary opposite nucleobases, each with at least one label, or at least two opposite bases offset by one base position relative to a complementary base pairing, each with at least one label.

8. A method according to any of the preceding claims, wherein in step d.-e. n different nucleic acid target sequences are indirectly detected by n different target sequence-independent reporter molecule types, wherein the detection of the signal generated in step d. takes place in k detection channels, where n > k and n > 2, and wherein at least two different target sequence-independent reporter molecule types are detected in step e. in the same detection channel and / or are represented in the same region of a data space in the representation under point e.

9. Method according to one of the preceding claims, wherein the signal of labeling a 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.

10. Method according to any of the preceding claims, wherein a target sequence-independent reporter molecule is an oligonucleotide or an oligonucleotide complex.

11. Method according to any of the preceding claims, wherein the nucleic acid detection reaction in step d. comprises an amplification method for DNA and / or cDNA.

12. Method according to the preceding claim, wherein the nucleic acid detection reaction in step d. is a PCR, RT-PCR, RPA or LAMP, and wherein, during DNA amplification, a mediator sequence of a mediator probe bound to a target nucleic acid is released by an enzymatic activity of a biomolecule, which then binds to a target sequence-independent reporter molecule, generating a signal.

13. Method according to one of the two preceding claims, wherein the detection under step e takes place within the framework of a digital amplification and / or signal generation.

14. Method according to any of the preceding claims, wherein the target sequence independent reporter molecules are universal reporters and / or modular reporter complexes and the at least one released mediator sequence is a component of a mediator probe PCR or mediator displacement LAMP.

15. Method according to one of the preceding claims, wherein at least one nucleic acid target sequence is derived from a conversion of another biomolecule into DNA sequence information.

16. Kit for carrying out the method according to any one of the preceding claims, comprising: at least one target sequence-independent reporter molecule of a first type comprising at least one label, at least one target sequence-independent reporter molecule of a second type comprising at least one label, at least one first mediator probe whose mediator sequence has an affinity for the at least one target sequence-independent reporter molecule of a first type and whose oligonucleotide sequence has an affinity for a first nucleic acid target sequence, at least one second mediator probe whose mediator sequence has an affinity for the at least one target sequence-independent reporter molecule of a second type and whose oligonucleotide sequence has an affinity for a second nucleic acid target sequence, optionally at least one buffer, optionally one polymerase, optionally one reverse transcriptase, optionally at least one PCR primer.

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