WO2016087637A1 - Method and system for detecting and distinguishing between at least two dyes - Google Patents

Abstract

The present invention relates to a method for distinguishing between the emission of light and/or the reflection of light from at least two dyes that are indistinguishable on the basis of their emission and/or reflection wavelength. The method involves at least two measurements being performed. Between the two measurements, the system is subjected to a treatment that alters the fluorescent intensity and/or luminous power of at least one of the dyes. The change of fluorescent intensity or change of luminous power that is characteristic of the dye and is brought about by the treatment between the measurements can be taken as a basis for identifying and/or quantifying the dye and/or the molecule marked thereby. The invention additionally relates to a system for carrying out the method.

Description

 METHOD AND SYSTEM FOR DETECTION AND DISTINCTION BETWEEN

AT LEAST TWO DYES

DESCRIPTION

 The present invention relates to a method for distinguishing between

Light emission and / or the light reflection of at least two dyes, which are indistinguishable on the basis of their emission and / or reflection wavelength. In the procedure at least two measurements are carried out. Between both measurements, a treatment acts on the system which alters the fluorescence intensity and / or luminosity of at least one of the dyes. Based on the characteristic of the dye

Fluorescence intensity change or luminosity change caused by the treatment between the measurements, the dye and / or the molecule labeled therewith can be identified and / or quantified. The invention further relates to a system for

Implementation of the procedure.

 BACKGROUND TO THE INVENTION

 In many biological, microbiological and chemical detection reactions are

Dyes and / or fluorescent dyes used to label analytes. The labeling is done to detect and distinguish the analytes. The detection of the analytes can be carried out qualitatively or quantitatively. For example, to differentiate analytes in a reaction or a test, various dyes are used. These dyes typically differ in their emission or reflection wavelengths. These differences are detected with a measuring device and a distinction is made between the different dyes and thus the different analytes. For example, DE 102008025328 B4 discloses a multiplex PCR method for the identification of HIV-1 and HIV-2 viruses. In this case, several probes are labeled with the same fluorescent dye pair, the fluorescent dyes having different excitation and emission wavelengths and thus can be separated via different color channels.

For many questions, it is necessary to be able to differentiate between many different analytes in an experiment, for example to save material or work, or to be able to achieve meaningful results at all. The possibilities of differentiating different dyes based on their characteristic emission or reflection wavelength are limited by the currently available standard devices. This raises the problem that it would be desirable to examine or discriminate more analytes than one has available detection channels. There are currently several ways to detect more analytes than there are colors. However, these methods have disadvantages, in some cases they can only be used for specific detection reactions. On the other hand, they often make demands on the

Boundary conditions under which a proof is to be carried out that can not always be fulfilled. The following methods of the prior art provide appropriate background

Information about the invention.

 "Barcodina"

 So-called 'barcoding' methods allow the distinction of more than one

Analyte (DNA molecule) per color channel if more than one color channel is used. To the

For example, in Huang et al (201 1, PLoS ONE 6 (1), p. E16033) various probes are used which are directed against different targets and labeled with a fluorescent dye. In contrast to common standard methods, not only one probe labeled with a dye of one color is used per target sequence. Instead, multiple probes are used which are directed against the same target sequence and with different ones

Dyes are marked. The ratio of these dyes to each other later in the detection allows conclusions about the presence or absence of different target sequences. The system is designed so that no combination occurs twice or can be represented as a mixture of other combinations. This will ensure that that

Presence of multiple target sequences makes a reliable distinction impossible. This procedure requires multiple probes per target sequence, significantly increasing the cost and complexity of the procedure. In addition, it is necessary to measure the intensity of the individual colors very reliably and to keep them apart. With the help of the system significantly more target sequences can be detected than color channels are available.

Multiplexing by template length

 Some known methods allow the discrimination of more than one analyte (DNA molecule) per color channel. For example, the method of Miotke et al. (2014, Anal. Chem., Pp. 140212095645005) uses intercalating dyes useful for the detection of

double-stranded DNA that was amplified in a digital droplet PCR. Two different target sequences were detected with two different PCR systems. The PCR systems are designed to produce two amplicons of different lengths (measured in base pairs). These different length amplicons are stained with an intercalating dye. The longer the amplicon, the more intercalating dye can intercalate into the double-stranded DNA. This increases the absolute fluorescence intensity for long amplicons over short amplicons. Thus, drops can be distinguished which contain no amplicons (very low

Fluorescence intensity) containing short amplicons (mean fluorescence intensity) containing long amplicons (high fluorescence intensity) and both long and short amplicons contained (very high fluorescence intensity). The system allows to detect more target sequences than colors are used. The use of intercalating dyes does not allow multiple colors to be used. In addition, the area to be amplified must meet certain criteria (here length) and absolute PCR efficiency is important to prevent misclassified samples. For example, lower PCR efficiency in extending a sample with a long target sequence could result in fewer amplicons being generated than in the short target sequence. This could cause the fluorescence intensity to be lower or comparable to that of such samples containing the short amplicons. This would falsely classify a sample with long amplicons as a sample with short amplicons.

 Combination of melting curves and colors for multiplexing

 This method allows the discrimination of more than one analyte (DNA molecule) per color channel. For example, the method according to Yiqun et al (2013, Nucleic Acids Res. 41 (7), p. E76) uses two different characteristics of the system, on the one hand different colors and on the other hand different melting points. By combining the two, a significantly higher number of target sequences are detected. The method uses melting curves, which must be included only for the differentiation of the analytes. In addition, a "linear after the exponential" PCR is used, which requires a more complex assay than conventional PCRs.

"Lifetime multiplexing"

 US6447724 describes a process with which different fluorescent dyes are distinguished by different long excited state lifetimes. The fluorescent dyes are used as markers. When a fluorescent dye is excited by light, the molecule is converted into an energetically higher, excited state. This condition has a certain lifetime, which is characteristic of the molecule. Although two different fluorescent dyes emit at the same wavelength (that is, the same color), they may differ in terms of excited state lifetime. These differences can be exploited to the dyes too

differ. However, it is necessary to measure the fluorescence intensity on a time scale well below a millisecond, since the lifetime of an excited state is in this range. Measurements with such a high temporal resolution are only possible with expensive and complex instruments, which are not standard equipment of laboratories for DNA detection.

 ..Multiplex digital PCR "

US20130178378 describes a method for increasing the degree of multiplexing in digital PCR so that more target sequences can be detected than colors are used. The principle is based on varying two different factors. The first factor of is changed is the amount of hydrolysis probe. It is believed that the hydrolysis probe is the limiting factor in signal rise during PCR. If a lot of probe is used for one target sequence and less used for another target sequence, it is assumed that the signal of the second target sequence lies below that of the first one. As a result, different targets can be distinguished by their endpoint fluorescence levels. The second factor that is varied is the percentage of the probe with

Fluorescent dye is labeled. If less probe marked with dye rises

Fluorescence intensity at a later time. This system requires the absolute efficiency of the PCR, otherwise the basic assumption that the probe is the limiting element for the fluorescence increase is no longer correct. In addition, more than one probe per target molecule is used.

 "Multiplex hybridization analysis" using color and melting temperature

 US6472156 describes a method for increasing the degree of multiplexing. Two probes hybridize to a DNA target sequence. Both probes are labeled with fluorophores that fluoresce by fluorescence resonance energy transfer (FRET). By measurements at different temperatures can be controlled, which of the probes still on the

Target sequence is hybridized and which has already resolved. The method is based exclusively on the analysis of the melting point of the probe with the target sequence and does not analyze the changed fluorescence behavior of the fluorophore at different temperatures. The method detects different loci with at least two different alleles. For multiplexing with standard DNA detection methods that do not meet this condition, the system does not seem appropriate.

 In light of the prior art, there are some disadvantages with the previously described methods.

Methods based on an unchanged PCR efficiency compared to comparative measurements are susceptible to PCR efficiency changes. However, these are not uncommon due to the use of different cyclers and readers used for PCRs. In addition, the system can only be transferred to other assays with increased effort, since verification tests must be carried out for each new assay to show that the efficiency of the PCR is high enough not to significantly affect the system.

 Methods based on a different length of the targets are in part disadvantageous, since the length of the target can not be freely selected in some cases, for example if particularly short DNA targets have to be detected.

 Methods using intercalating dyes are not suited to use different colors to increase the multiplexing degree, since the reaction of the

intercalating dye is sequence-unspecific. This reduces the level of multiplexing of a complete system by a factor of 4-6 over other methods. Methods that use the excited state lifetime of fluorescent dyes to distinguish the dyes require very expensive measurement setups. In order to differentiate the dyes reliably, temporal resolutions far below milliseconds are necessary. Such devices are expensive and are not standard on DNA detection laboratories.

 Methods based on an unchanged PCR efficiency compared to comparative measurements are susceptible to PCR efficiency changes. However, these are not uncommon due to different cyclers and readers used for PCRs. In addition, the system can only be transferred to other assays with increased effort, since verification tests must be carried out for each new assay to show that the efficiency of the PCR is high enough not to significantly affect the system.

 Methods that specialize in the detection of at least two alleles are not suitable for many detection reactions and therefore not universally applicable.

 This results in the need for a method of distinguishing dyes, which is not based on the emission or reflection characteristics, is to be used as universally as possible and can be integrated into existing methods and devices with little additional effort.

 Furthermore, a method for the quantitative determination of the content of aggregates of substances in mixtures is disclosed in WO2003 / 054548 A1. The application of different fluorescently labeled protein-based probes that your

Change detection properties in the aggregate state. In DE 102012203964 B3 is a

Method for determining the concentration of nucleic acids described. This involves the use of two nanoparticle-based probes, which show a measurable change in their detection properties when they are in close proximity to each other.

 DE102008019756 B4 discloses a method for measuring velocities in a fluid flow, in which marked particles with different dyes are used which have measurable changes in the detection properties as a function of the printer or the temperature. With the method, the temperature or

Pressure distribution in flows are measured. Likewise, from EP 09190604 B1, DE1589382 and WO201 1018355 A1 various dyes are known which your

Change properties under different conditions. Thus, EP 09190604 B1 describes color change materials whose color changes as a result of the action of heat or water. DE 1589382 discloses a luminescent substance whose luminescence can be changed with the aid of a magnetic field. WO2011018355 A1 discloses pressure sensitive inks. Such processes disclose dyes which, under particular conditions, can exhibit different emission and thus detection properties. Nevertheless, dyes of this type have not yet been effectively and selectively combined to allow detection of multiple target molecules in a color channel. DETAILED DESCRIPTION OF THE INVENTION

 In the light of the prior art, it was therefore an object of the invention to provide a means which allows the identification of several dyes and / or the discrimination of several dyes, without having the disadvantages of the prior art.

 The problem of the invention is solved by providing a method for discriminating between the light emission and / or the light reflection of at least two dyes that are not available based on their emission and / or reflection wavelength

are different, comprising the steps:

a) providing the at least two dyes which are indistinguishable on the basis of their emission and / or reflection wavelength and are preferably present as a mixture in a sample;

b) treating the at least two dyes, wherein the treatment causes a change in the light emission and / or the light reflection of at least one of the dyes; and

c) measuring the combined light emission and / or the light reflection of the at least two dyes, at least before and after the treatment in step b);

characterized in that

d) the at least two dyes are differently changed by step b) with respect to their light emission and / or light reflection, and

e) the concentration of at least one of the dyes by the modification of

 measured light emission and / or light reflection between the treatments in step c) is calculated.

 The present invention therefore relates to methods of distinguishing two or more dyes from each other. The two dyes emit or reflect light of a similar or equal wavelength (i.e., they are substantially the same color). The two dyes can not be distinguished with a conventional fluorescence detector or by conventional colorimetric measurement.

 The term "at least two dyes based on their emission and / or

Reflect reflection wavelength "refers to two dyes whose emission and / or reflection wavelength is so close to each other that they are indistinguishable from classical analyzers based on bandpass filters, for example Based on their emission and / or

Therefore, the term preferably also refers to the analyzer to be used, the term therefore also includes at least two dyes, the based on their emission and / or reflection wavelength with the applicable

Analyzer (eg in a detection channel) are indistinguishable.

 In a preferred embodiment it is therefore provided that the at least two

Dyes, "not based on their emission and / or reflection wavelength

"dyes are those whose emission and / or reflection wavelength may be different, but are substantially less different than the bandwidth (detection channel) used for detection." This means that the two dyes have emission and / or or have reflection wavelengths that are so close to each other that both can be detected by a single detection channel used in the method, eg, that both dyes can be detected in a single color channel A color channel or detection channel is characterized by a wavelength range in which Measuring a device without it (in terms of wavelengths) finer resolution.

 In one embodiment, it is therefore provided that the at least two dyes "which are indistinguishable on the basis of their emission and / or reflection wavelength" are dyes whose emission and / or reflection wavelengths have less than 100 nm difference from one another, preferably 90 nm , 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 1 nm or less than 1 nm difference from each other.

 Traditional bandwidths vary from device to device and also differ in terms of color channels. Most conventional bandwidths are in a range of, for example, 10 nm to 40 nm.

 The method is preferably characterized in that the at least two dyes "which are indistinguishable on the basis of their emission and / or reflection wavelength" are dyes having different properties with respect to their

Fluorescence intensity change or luminosity change after treatment in step b) have. The calculation of the concentration of a dye according to step e) comprises all possible calculations which are suitable for determining absolute and / or relative

To determine concentrations of one or more of the dyes. The calculation is not limited to the fact that absolute concentration levels of a dye (or associated target molecule) must be established. The calculation of the concentration includes, for example, the preparation of a statement that Dye A (or the associated target molecule A) is present in larger amounts, and / or to a greater extent

Total fluorescence contributes, as dye B (or the associated target molecule B). Thus, one possible result of calculating the relative concentration of Dye A to Dye B is a binary value that states whether Dye A or Dye B is present in greater amounts without having to determine the quotient of the levels of concentration. The determination of the mere presence of a target molecule based on the "calculation" of a dye - without providing specific concentration data - is also included in step e). The calculation of the concentration of a dye involves calculating the concentration of an unbranched dye if quenchers are used in the process. It may be that the absolute dye concentrations in the process are known and that the result of the process depends on the signal of an unbranched dye. It is preferred that the concentration of the uncquenched dye correlate with a concentration of a biological target molecule. A target molecule in the sense of the invention is a molecule which is to be detected or quantified by the method according to the invention. The method can be used to detect and / or quantify several target molecules. The calculation of the concentration of a dye according to step e) thus likewise comprises a calculation of the (absolute and / or relative) concentration of

Target molecule. In this case, the dye during the process steps a) - d) on the

Target molecule present. For example, the dye may be bound to a probe along with a quencher, whereby quenching is abolished by the binding of the probe to the target molecule. However, it may also be that the dye is not bound to the target molecule during process steps a) to d). In particular, it may be preferred that a probe is used with a dye and a quencher, wherein the quenching is abolished by cleavage of the dye from the probe after it binds to a target molecule. In this case it may be preferred that the binding of the probe to the target molecule and the cleavage of the dye before the

Process steps a) to d) takes place. This is the case, for example, when using TaqMan probes in a PCR amplification method. In this case, the determination of the concentration in step e) of the dye cleaved off from the probe (and thus the undquenched dye) allows a determination of the concentration of the target molecule.

 The calculation of the concentration of a dye according to step e) comprises computer-implemented methods or approaches that can be used in performing step e). The invention therefore, in another embodiment, relates to software configured to perform such a calculation. Such software can be in one

Embodiment of the invention comprise further component or modules which are suitable for the

Implementation, control or evaluation of a PCR or automated ELISA method are suitable. Such other software components and modules may be identified by one skilled in the art of molecular analytical technique.

 The at least two dyes can label different molecules by being associated with or bound to these molecules. The two dyes can be fluorescent dyes. They emit or reflect light of a similar or the same wavelength. Therefore, they can not be distinguished by their fluorescence wavelength with conventional analyzers in a detection channel.

In the procedure at least two measurements are carried out. Between both measurements, a treatment on the system, which affects the fluorescence intensity or luminosity changed. In a preferred embodiment of the invention, the term "treatment which causes a change in the light emission and / or the light reflection of at least one of the dyes" encompasses all possible treatments which produce the desired effect, preferred are treatments which are to be understood as external treatments "External treatments" refer to changes in the physical conditions of the sample to be analyzed, wherein the external treatment does not change the content, components and / or components of the sample, or the number of components of the sample, e.g. B. if no additional new components are added in the mixture from the outside, for example, when the temperature is changed or a light irradiation is given without opening the sample container.

 Examples of treatments under b) are irradiation with light (the excitation wavelength), temperature changes, Red / Ox potential changes, treatment with electromagnetic fields, pH changes, surface changes or pressure changes. The change in fluorescence intensity or luminosity is reversible, irreversible or partially reversible. Based on the characteristic of the dye fluorescence intensity change or

 Luminosity change caused by the treatment between the measurements, the dye and / or the molecule labeled therewith can be identified.

 The method according to the invention is therefore characterized in one embodiment in that the treatment in step 1 b) comprises a light irradiation, preferably with a corresponding excitation wavelength, a temperature change, a Red / Ox potential change, an electromagnetic field change, a pH change, a surface modification and / or includes a pressure change.

 In a preferred embodiment, therefore, the invention relates to a method as described herein, characterized in that the treatment in step b) leads to a different reduction of the intensity of the light emission and / or the light reflection of the respective dyes. FIGS. 1 and 2 show examples of the different changes in the fluorescence intensity or luminosity.

In a preferred embodiment, the method is characterized in that the treatment in step b) is a light irradiation and this leads to a greater reduction in light emission and / or light reflection in a first of the at least two dyes than in a second of the at least two dyes and the calculation of the concentration in step e) by comparing the quotient of the combined light emission measured in step c) and / or the light reflection of the at least two dyes after and before the light irradiation (L _na c _h / L _V or) with one or more Thresholds occur, with

a) in the case that the quotient L _na c _h / L _V or greater than a first threshold, a higher concentration of the second dye compared to the first dye is present and

b) in the case that the quotient Lnac _h / or is less than a second threshold, there is a higher concentration of the first dye compared to the second dye.

It may be preferred that the first and second threshold values are identical and that the method has exactly one threshold value. In that case, the result of the calculation is preferably a binary result which provides information as to whether a first dye (and preferably a first target molecule associated therewith) or a second dye (and preferably a second target molecule associated therewith) is present.

It may also be preferred that the first threshold be greater than the second threshold, and in the case that the quotient L _to / L _be greater than or equal to the second threshold

 Threshold and less than the first threshold, the concentration of the first colorant is indistinguishable from the concentration of the second colorant. Here, the result of the calculation in step e) can be an indication as to whether the first dye (and preferably a first target molecule associated therewith), the second dye (and preferably a second target molecule associated therewith) or that both dyes are present. By providing such an "intermediate region" for which no statement is made as to which dye is present in a higher concentration, advantageously the safety of the method for the detection of target molecules is increased.

 The method according to the invention is therefore characterized in one embodiment in that the change in the light emission and / or the light reflection through step b) is irreversible. Such treatments may be carried out, for example, when the dyes need not be differentiated a second or repeated time. Such an analysis can be carried out, for example, if the dyes are already attached to their target molecules and the presence and / or quantity of the respective targets are to be determined once.

 The method according to the invention is characterized by a further embodiment

characterized in that the change of the light emission and / or the light reflection by step b) is reversible or partially reversible, in particular in a method for the determination of the dyes in a "real-time" assay, eg RT-PCR are suitable for use in a detection process in which further detection steps are required in the later process.

In one embodiment, the process according to the invention is characterized in that the dyes are fluorescence dyes. These fluorescence dyes are preferably selected from the following list: cyanines, fluoresceins, rhodamines, Alexa fluors, Dylight fluors, ATTO dyes, BODIPY dyes, SETA dyes, SeTau dyes, especially Alexa Fluor 350, DY-415, ATTO 425, ATTO 465, Bodipy FL , Alexa Fluor 488, FAM, FITC, fluorescein, Fluorescein-dT, ATTO 488, Oregon Green 488, Oregon Green 514, Rhodamine Green, TET, ATTO 520, JOE, Yakima Yellow, Bodipy 530/550, HEX, Alexa Fluor 555, DY-549, Bodipy TMR-X, Cyanine 300 ATTO 550, TAMRA, Rhodamine Red, ATTO 565, ROX, Texas Red, Cyanine 350, LightCycler 610, ATTO 594, DY-480 XL, DY-610, ATTO 610, LightCycler 640, Bodipy 630/650, ATTO 633, Alexa Fluor 647, Bodipy 650/665, ATTO 647, ATTO 647N, Cyanine 500, DY-649,

LightCycler 670, Cyanine 550, ATTO 680, LightCycler 705, DY-682, ATTO 700, ATTO 740, DY-782, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7, Pyrene, Dansyl-X, Eterneon 350/430, Eterneon 350/455, Dy 350, Aterneon 384/480, Atto 390, Eterneon 393/523, Eterneon 394/507, Dy 405, Alexa Fluor 405, Oyster 405, HYCO, Dy 415, DyQ425, D-AMCA, Alexa Fluor 430, Atto425, Abberior STAR440SX, Atto 465, Abberior STAR 470SX, Dabcyl, Eterneon 480/635, Dy 485 XL, Chrmoeo 488, Oyster 488, Dy 490, Fluorsecein 5-ΕΧ, BHQ 0, FAM, Dy 495, Chrmoeo 494, Alexa Fluor 488, Atto 495, Ibapy 946/503, Atto 488, Abberior STAR 488, IBApy FL, Alexa Fluor 500, DyQ 505, Dy 480 XL, Rhodamine 110X, Dy 505, Dy 510 XL, Atto 514, Abberior STAR 512, Atto 520, Alexa Fluor 514, Dy 481 XL, JOE 6-isomer, TET, Dy 520 XL.

Carboxy Rhodamie 6G 6-isomer, Carboxy Rhodamie 6G 5-isomer, Dy521 XL, IBApy R6G, Alexa

Fluor 532, Atto 532, Dy 530, IBApy 530/550, BHQ-1, Atto Rho6G, HEX, Atto 540Q, DyQ 1, IBApy TMR-X, Dy 554, TAMRA 5-isomer, Dy 555, Quasar 570, Dy 556 , Chromeo 546, Oyter 555, Oyster 550, Atto 550, Alexa Fluor 555, Cy3, TAMRA 6 isomer, Alexa Fluor 546, Dy 548, Dy 547, Dy 560, Dy 550, Dy 549, Dy 549P1, Atto 565, Atto Rho3B, Oyster 568, Atto Rho1 1, Atto Rho12, Alexa Fluor 568, BHQ-2, Atto Thio 12, Dy 591, Dy 590, Abberior STAR 580, Sulforhodamine 101, Atto 580Q, Atto Rho101, Alexa Fluor 594, Cy3.5 , Dy 594, Oyster 594, Atto 590, BBQ 650, Dy 605, Atto Rho 13, Atto 594, Alexa Fluor 610-X, Dy 610, Alexa Fluor 610, Atto 610, Atto 612Q, Atto 620, Dy 615, Atto Rho14 , Atto 633, Alexa Fluor 633, Alexa Fluor 635, Abberior STAR 635, Abberior STAR 635P, EVOblue ™ 30, Dy 634, Dy 632, Dy 631, Dy 630, Dy 633, Atto 647N, Quasar 670, Atto 647N, Cy5, Chromeo ™ 642 , Dy 636, Oyster 647, Dy 635, Alexa Fluor 647,

Oyster 650, Dy 647, Dy 652, Dy 654, Dy 649P1, Dy 648, Dy 651, Dy 650, Dy 649, Atto MB2, DyQ 660, DyQ 6616, Atto 655, Atto Oxa 12, Atto 665, Alexa Fluor 660, Methylene Blue, BHQ-3, Cy5.5, Dy 677, Dy 678, Dy 675, Dy 676, Oyster 680, Dy 679P1, Dy 679, Alexa Fluor 680, Atti 680, DyQ 3, Dy 680, Dy 682, Dy 681, DyQ 700, Atto 700, Alexa Fluor 700, Dy 703, Dy 704, Dy 700, Dy 701, Atto 725, Dy 734, Dy 730, Dy 732, Dy 731, Atto 740, Oyster 750, Dy 754, Alexa Fluor 750, Dy 752, Cy7, Dy 750, Dy 751, Dy749P1, DyQ 4, Dy 778, Dy 777, Dy 776, Dy 780, Dy 781, Dy 782, Alexa Fluor 790, Dy 831.

 One of ordinary skill in the art will be able to select combinations of dyes that are useful as dyes of the present invention based on their emission and / or

Reflection wavelength are indistinguishable, can be used. This selection depends on the device to be used, in particular on the detection channels and their bandwidth, which are to be used for the combined detection of the signal of the respective dyes. An average expert is also capable of

To determine fluorescence intensity change or luminance change of a dye caused by the treatment between the measurements. In one embodiment, the method according to the invention is characterized in that at least one of the dyes is in spatial proximity to a quencher so that the measured light emission and / or the light reflection of the at least two dyes results from the sum of the unquenched components of the dyes Efficiency of the quencher is changed by step b), and when two or more quenchers are in spatial proximity to the at least two dyes, the efficiency of the respective quenchers is changed differently by step b).

 This embodiment is therefore relevant, as well as quencher mentioned by the

Treatments (temperature, light, etc.) change their quenching efficiency. So it might also be possible to use two identical dyes with different quenchers and thereby distinguish two different TaqMan probes. Thus, the following embodiment of the invention is also encompassed: when, for example, a probe with quencherl and fluorophore is used and a second probe 2 with quencher 2 and fluorophore 2 is used. Now the quenching efficiency of Quencherl decreases due to the treatment, the

Fluorescence intensity of fluorophore increases. This leads to an increased fluorescence signal from Sondel. The quenching efficiency of Quencher2 increases due to the treatment

Fluorescence intensity of fluorophore2 decreases. This leads to an increased fluorescence signal from probe2. Now you can tell by the direction of the change (rising or falling) which probe it is.

In particular, it also means "dark quencher", which absorbs light and thus light energy, but does not or only partially emit at other wavelengths.These dark quencher give off the absorbed light energy in other forms of energy, eg heat the example below explained in more detail.

In a preferred embodiment of the invention, the method is characterized in that the light emission and / or the light reflection of the dyes correlates with the concentration of biological molecules. By the term "biological molecules" is meant preferably carbonaceous molecules, including macromolecules, found in a biological source, as well as derivatives, analogs and modifications of such molecules. In addition, the term refers to carbonaceous molecules such as drugs, antibiotics and the like used in medicine. The term also refers to

various other biologically significant carbonaceous molecules, such as toxins, pesticides and herbicides, that can be studied in medicine or environmental testing. For example, nucleic acid analogs such as morpholino oligos containing modified bases that are not found in nature are also referred to as biological molecules. Accordingly, all analogues of a molecule in nature or any chemical modification of such a molecule are also preferably included in the definition of biological molecules. Biological molecules can be isolated from natural sources or synthesized in the laboratory, as is For example, the case is for synthetic peptides or oligonucleotides. In this preferred embodiment, the method can be used to detect biological target molecules.

 It may be preferred that the dyes during the execution of the

bound biological molecules present. For example, it may be preferable that the

Dyes bound to a probe and the light emission of the dye is increased when the probe binds to a biological target molecule. Such a probe may for example be a molecular beacon. Molecular beacons are hybridization probes for the detection of DNA consisting of a single-stranded DNA with a hairpin structure, with one dye and one quencher each at a 3 'or 5' end of the DNA strain.

By binding a molecular beacon to a complementary DNA target, the distance between the quencher and the dye increases. The increased distance leads to an increase in the light emission of the dye. Thus, the light emission of the dye correlates with a concentration of the DNA target molecule, since it can be deduced from the fluorescence emission of the dye conclusions about the presence of the DNA target molecule. In that case, while the method steps a) to d) are carried out, the dye is bound to the biological molecule to be detected.

 However, it may also be preferred that the dye be used during the performance of the

Process steps a) to d) are not bound to the biological target molecule. For example, the principle of detection of TaqMan probes is based on that release of the dye from the probe indicates the presence of target biological molecules. TaqMan probes are hybridization probes, comprising a dye and a quencher, for the detection of DNA target molecules during a PCR amplification reaction. In the unbound state of the probe or in the bound state of the probe to the complementary DNA target molecule, the light emission of the dye is suppressed by the quencher. Due to the polymerase activity during amplification of the target DNA molecule, the fluorophore is cleaved from the portion of the probe comprising the quencher and the light emission of the unbound, uncquenched dye is increased. Although during the execution of the method steps a) to d) the dye is not bound to the biological target molecule, the light emission of the dye correlates with the concentration of the DNA target molecules. It may thus also be preferred that in one embodiment of the invention the dyes are at least temporarily bound to biological molecules, wherein the temporary bonding has taken place before step a) of the process.

 The method according to the invention is characterized in a preferred embodiment

in that the dyes are bound to biological molecules, preferably nucleic acid, antibodies and / or antigens. Accordingly, biological target molecules can be detected. The invention provides techniques for detecting multiple biological molecules in a sample, wherein more molecules than available color channels or detection channels can be detected. Another aspect of the invention therefore relates to the use of the invention

Method for the determination of two or more nucleic acids, for example

Nucleic acid products of an amplification reaction, preferably polymerase chain reaction (PCR).

In a preferred embodiment, the invention relates to the use of the method for the determination of two or more nucleic acids, wherein the dyes are bound to different oligonucleotides and the oligonucleotides allow a sequence-specific binding to one or more target sequences, wherein the dye-oligonucleotide combinations preferably from the TaqMan probes, molecular beacons, hybridization probes, RPA probes, mediator probes, FRET probes and / or Q _G probes.

 Another aspect of the invention relates to the use of the method according to the invention for the determination of two or more protein targets, preferably antibodies or antigens, preferably in an immune or cell assay. A particularly preferred immunoassay is the enzyme-linked immunosorbent assay (ELISA).

 A further aspect of the invention relates to a system for distinguishing between the light emission and / or the light reflection of at least two dyes, which are indistinguishable on the basis of their emission and / or reflection wavelength, comprising:

a) at least two dyes which are (i) based on their emission and / or

 (Ii) show different changes in their light emission and / or light reflection, which are caused by the treatment according to claim 1 b), and (iii) are present as a mixture in a sample;

b) means for treating the at least two dyes, wherein the treatment is a

 Causing change in the light emission and / or light reflection of at least one of the dyes,

c) means for measuring the light emission and / or the light reflection of the at least two dyes, for. B. a fluorescence and / or colorimetric detector.

 The system according to the invention is preferably characterized in that means for irradiating the at least two dyes, e.g. Light sources, preferably a light emitting diode (LED) or mercury vapor lamps or other lighting elements, are present in the system.

Further agents for the treatment of the dyes may be heating and / or cooling elements which can change the temperature of the sample containing the dyes in a directed manner. Further agents for the treatment of the dyes may be pH-changing substances (eg acids or alkalis) which are added to the sample or delayed their pH-altering effect or released by an external action (eg encapsulated acids or alkalis and / or so-called "caged protons ", chemical compounds that change their structure upon irradiation with light, releasing a proton and lowering the pH of the solution.) Agents for the treatment of the dyes may be electromagnetic coils that can selectively expose the sample to an electromagnetic field.

 The invention can be used in diagnostic procedures in which the identification of particular biological molecules can provide disease-relevant information. The inventive method is also conceivable in all analytical applications, where different molecules must be identified. The invention enables analytical or diagnostic statements that were previously possible only with very complex devices or costly procedures.

 By the method according to the invention, multiplex methods, basically all types of detection of several analytes in a sample, can be reduced

Number of detection channels can be simplified. This reduces the requirements for the device to be used for the method. Devices that were not previously suitable for a detailed multiplexing process can now be used effectively with application of the invention.

By the method according to the invention more analytes can be detected in a reaction solution with the same device than before. This reduces the material and

Personnel costs significantly and increases efficiency.

 Possible applications of the invention:

 Application for DNA detection:

Possible applications of the invention are based on the finding that the change in the fluorescence intensity of fluorescent dyes, which is caused by external changes, such as irradiation with light of the excitation wavelength or changes in temperature, is sufficiently large to be measured with conventional, inexpensive laboratory equipment. The differences are also great enough to distinguish two different commercially available dyes with high security.

In other examples, the dyes to be distinguished are linked to oligos that serve to detect DNA. These oligos may be, for example, one of the following probes: TaqMan probes, molecular beacons, hybridization probes, RPA probes, mediator probes, FRET probes or Q _G probes.

The detection of the DNA may e.g. by means of PCR or isothermal methods, but also via hybridization of probes.

 Applications that exploit the bleaching behavior of dyes:

In other examples, multiple fluorescent dyes can be distinguished by irradiation with light. For example, the light can come from a commercial LED, which is available at low cost. The light can, for example, from the excitation light source of the readout unit come. The light should be (at least partially) in the excitation spectrum of at least one of the two dyes. The fluorescence intensity is measured before and after the irradiation with light. One of the two dyes bleaches by irradiation with light stronger than the other. A second measurement of fluorescence intensity is made after irradiation with light. Now the measured values are compared. On the basis of the percentage decrease, conclusions can be drawn as to which of the two dyes is present in the sample or contributes more strongly to the fluorescence signal. FIG. 3 shows a schematic illustration of the method.

 Applications that exploit the temperature dependency of dyes:

In further embodiments, the two fluorescent dyes are distinguished by changing the temperature. The temperature is e.g. during a PCR routinely changed so that a temperature change of the sample is easy to implement. It will be the

Fluorescence intensity measured before and after changing the temperature. One of the two dyes changes its fluorescence intensity more than the other when the temperature is changed. A second measurement of fluorescence intensity is made after the change in temperature. Now the measured values are compared. On the basis of the percentage decrease, conclusions can be drawn as to which of the two dyes is present in the sample or contributes more strongly to the fluorescence signal. A schematic representation of the principle is shown in FIG.

Applications that exploit changes in quenching efficiency:

 In further examples, the change in quenching efficiency of the quencher is exploited to distinguish a DNA detection probe. Many DNA detection probes consist of a fluorophore and a quencher that is brought close to the fluorophore via a DNA oligo. As long as the fluorophore and quencher are in close proximity, most of the fluorescence emission is quenched by the quencher and is quenched as heat or light from others

Wavelength delivered to the environment. During the detection reaction, in the presence of analyte, the quencher is spatially further removed from the fluorophore and the fluorescence intensity of the fluorophore increases. If the quenching efficiency of the quencher changes, so does the overall fluorescence of the system. Through a clever selection of fluorophore and quencher, it is possible to show which probes the quencher spatially differs from

 Fluorophore was separated and in which not. The change in quenching efficiency may be e.g. caused by light (e.g., the excitation wavelength),

Temperature changes, Red / Ox potential changes, electromagnetic fields, pH changes, surface changes or pressure changes. The change in quenching efficiency is reversible, irreversible or partially reversible.

Application in digital PCR: In further examples, the inventive method is used to the

Multiplexing degree of DNA detection reactions of various kinds increase. This can e.g. digital detection reactions such as digital PCR or digital isothermal detection methods.

Application in immunoassavs:

In further examples, the inventive method is used to the

 Increase the level of multiplexing of immunoassays of various kinds. In this case, e.g. the analyte, antigen or antibody is labeled with dye. In addition, a dye, e.g. be released by an enzyme reaction.

 Application in cell markers:

In other examples, the technique of differentiating two dyes is used to distinguish different cell markers. The term "cluster of differentiation" (CD), for example, refers to groups of immunophenotypic surface features of cells which can be classified according to biochemical or functional criteria. Because different CD molecules, or others

Cell markers specific for a particular variety or stage of development of cells may be used as markers that can be recognized by antibodies coupled with dyes and thus detected.

 Application in combination with Quantum Dots:

 In further examples, the particular properties of Quantum Dots (QD) or related dyes are exploited. Compared to conventional fluorescent dyes, QD is exceptionally stable to fading. Thus, QD dyes and conventional fluorescent dyes emitting at the same wavelength can be distinguished. One possible application is shown schematically in FIG.

 Application in combination with Molecular Beacons:

Molecular Beacons (MB), which after a successful amplification open to a DNA

 Template attached, generate a fluorescent signal, which is increased compared to a time before the amplification of the target. This fluorescence level decreases with increased temperature as the MB melts and collapses again. As the temperature further increases, the "star" of the MB melts and the fluorescence signal increases again, and this behavior can be exploited by designing different MBs

have different melting points. To make these melting points independent of the sequence to be detected, a system can be used which contains a primer containing a generic to MB reverse-complementary sequence. Measurements at different temperatures now allow conclusions about which target sequence, and associated with it, which generic primer sequence was amplified. Also one

Combination of MB and hydrolysis probes is conceivable. By reading at 3 different temperatures can thus be distinguished more than 2 target sequences per color. All probes may be labeled with dyes that emit at the same or a very similar wavelength. Possible applications are shown schematically in FIG. 6 and FIG.

Advantages of the invention over prior art:

 An advantage of the system is to increase the degree of multiplexing to> 1 per color without imposing additional restrictions on the system. This is of great importance, especially with regard to DNA detection, because a method is the more universally applicable the less

Conditions to the target sequence to be detected.

Unlike other methods, increasing the level of multiplexing for the method shown here does not require designing more than one probe per target sequence.

 In contrast to other methods, in the method presented here one is free to choose the target sequence. This also applies in particular to the length of the target sequence, which is not subject to any further conditions.

In contrast to other methods, the method presented here does not depend on the absolute efficiency of a detection reaction. Because the values before a change are compared to values for changes, the course of any upstream reactions or changes (such as amplification, e.g., by PCR) is not relevant to the detection method.

In contrast to other methods, the method presented here requires at least two measurements, so it can be implemented with less effort than other methods.

 Unlike other methods, there is no need for the method shown here

elaborate measurements with a high resolution of time or wavelength can be performed.

In contrast to other methods, the method shown here can be implemented with conventional analyzers and requires no additional handling effort for some embodiments. For example, "conventional analyzers" may be the following real-time cyclers: RotorGene6000, RotorGeneQ, LightCycler96, Mx3000P QPCR System, qTOWER Real-Time Thermal Cycler, TOptical Real Time Thermal Cyclers, CFX Connect Real-Time PCR Detection System, CFX384 Touch ™ Real-time PCR detection

Systems, CFX96 Touch Deep Well® Real-Time PCR Detection System, CFX96 Touch Real-Time PCR Detection System, Quantstudio ™ 12K Flex Real-time PCR System, StepOnePlus ™ Real-Time PCR System.

CHARACTERS In the following, the invention will be explained in more detail with reference to the following figures, without being limited to these.

 Fig. 1: Left: The two dyes bleach out at different speeds. Based on

 Remaining residual fluorescence, the two dyes can be well distinguished. On the left for the pair FAM / Atto488, both fluoresce on a similar wavelength, right for the pair Cy5 / Atto647N, both fluoresce at a similar wavelength. Both FAM and Cy5 are common dyes in DNA analysis that can be read by standard equipment.

 Fig. 2: Falschfarbenfluoreszenzaufname a Droplets (diameter about Ι ΟΟμιη). Between the measurements, the droplet is exposed to the light of the excitation wavelength in the device (white numbers: minutes after the start of the measurement). Clearly visible is the fading of the droplet. This shows that the principle is also applicable in digital PCR.

 Fig. 3: Schematic drawing of the sequence of a PCR in which the system presented here is used. During and after the PCR, the two targets can not be distinguished, since they are labeled with two dyes that fluoresce on a similar wavelength (the different degrees of increase and the different "cycle of quantification" value are purely exemplary and can also be the same) By bleaching the dyes both dyes lose their different speeds

 Fluorescence intensity. Now let the dyes and thus the targets

 differ.

 Fig. 4: Schematic representation of the principle presented here. The curves show the course of the fluorescence intensity with increasing temperature after the PCR. Sample 1

 (dashed curve) loses its fluorescence intensity much faster than sample 2 (dotted curve). Although both dyes fluoresce on a similar wavelength they can now be distinguished.

 Fig. 5 Schematic representation of the principle set forth here. The curves show the course of the fluorescence intensity under irradiation with light after the PCR. Sample 1 (dashed curve) loses its fluorescence intensity much faster than sample 2 (dotted curve), which in turn loses its fluorescence intensity faster than sample 3

 (solid line). Here are sample 1 and 2 with conventional

 Fluorescent dyes labeled, sample 3, however, with QD, which compared to a

 Fading is extremely stable. Although all three dyes on a similar

 Fluorescence wavelength can now be distinguished.

 FIG. 6: Two systems are shown target molecules with a molecular beacon (MB)

prove. In this case, the reverse complementary sequence of the MB is contained as a linker on the primer. By PCR or a similar amplification reaction, the Target molecules multiplied. If there is more target molecule, the MBs will fold up and therefore increase the fluorescence level. In this case, the sequence of the left MB complementary to the primer on the left is shorter than that of the right one.

 Fig. 7: Left: Four different cases are listed, each containing different combinations of the two target molecules or neither. Two measurements at 55 ° C and

70 ° C are carried out the fictitious results of the fluorescence measurement are for each of the four cases right of the temperatures. The different length of the hybridization sequences of the MB requires different melting temperatures. These are used to distinguish all 4 cases. Right: Two melting curves for different target sequences and the measurements with their results.

 Fig. 8: The time in minutes is shown on the x-axis. On the y-axis, the fluorescence intensity normalized to t = 0 is shown in arbitrary units. Red crosses reflect the fluorescence intensity of the sample spiked with E. coli DNA. Black squares represent the fluorescence intensity of the sample spiked with B. subtilis DNA.

 Fig. 9: Example of the temperature behavior of FAM & Atto488. Black squares (top) correspond to only the Cy5 target sequence present, circles (medium) correspond to Atto647N target sequence only, triangles (bottom) correspond to both target sequences present.

Fig. 10: Differences in the fading of PCR samples after completion of the PCR. In each color channel two different targets can be distinguished. These are in red

Color channel mecA and mecC and in the green color channel nuc and PVL. For each of the color channels were three different combinations of the target DNA with a

Concentration of 10 ⁵ cp. μ ^{1 was added} to the PCR sample. As can be seen, all three combinations can be distinguished with a confidence level greater than 99%. The data points represent inter- and intra-experimental variability. For each

Data point 3 experiments were performed with 4 replicates.

Fig. 11: Experimental data for the remaining fluorescence of PCR samples comprising fluorescent samples for which different dyes were activated. For this, the fluorescence after bleaching for 6 minutes under irradiation of white light is compared to the initial fluorescence before bleaching. All dyes were present at a concentration of 200 nM, only the Cy5 labeled sample was used at a concentration of 20 nM. The DNA concentration was 10 ⁵ cp in all cases. μΓ ¹

 FIG. 12: Level of confidence with which the shown combinations of targets can be distinguished from experiments in which only one target has been amplified. In all

Experiments, the same 4-plex PCR experiment was used according to Example 5. Each data point represents 4 replicates. Fig. 13: Quantification of DNA target sequences using monochrome multiplexing in droplet digital PCR (ddPCR). A) 4 samples with different concentrations of E. coli DNA (220, 440, 880, 1760 cp. ΜΙ ^"1 ) and constant concentration of ß. Subitlis DNA (200 cp. ΜΓ ¹ ) were amplified together by amplification of E coli DNA, the sample labeled with the slow bleaching Atto488 is desquenched

 "Unquenching" refers to the increase in fluorescence of the labeled sample as soon as it binds to DNA, since this reduces the influence of a quencher, resulting in an increase in the proportion of undquenched or uncquenched dyes. Subtilis DNA, on the other hand, is entquenched with the rapidly bleaching FAM labeled sample

 Fluorophores, FAM and Atto488, emit light of comparable wavelength and were distinguished by a monochrome multiplexing process. (A) The abscissa shows concentration of the initial E. coli DNA copies, and the ordinate shows concentration of E. coli DNA measured in 4 digital PCR experiments after fading. (B) The same procedure was for three different ones

Concentrations of ß. subitilis DNA (220, 632, 2000 cp. μΓ ¹ ) and a constant concentration of E. coli DNA (600 cp. μΓ ¹ ) repeated. For each of the above experiments, 150 drops were evaluated.

 Fig. 14: Decay of fluorescence intensity in droplet digital PCR (ddPCR). It will be the

 normalized decay curve of the fluorescence intensity of over 200 positive drops

3 different experiments (for each dye except for "Atto488 and FAM": -50 drops from 3 experiments) containing FAM and Atto488 labeled samples, with the one named in the legend being desquenched, using the light source of the scanner for fading ,

Fig. 15: Photobleaching in droplet digital PCR. A) shows a microscope image of the drops before and after 10 minutes of irradiation with white LED light. The light drops in the "before bleaching" pictures contain FAM quenched dye

Visualization are some marked with arrows. All bright drops in the "after-fade" images contain Atto488 de-quenched hydrolysis samples, as the FAM fluorescence intensity is lowered to a background level, as can be seen on the drops, which are marked with an arrow The numbers in the upper left corner of the "Before bleaching" images correspond to the expected number of E. coli targets per μΙ). Each image drops the drops after the digital droplet PCR in the readout chip. All drops contain the same PCR mastermix, the target sequence of the DNA was randomly distributed. B) shows the decaying fluorescence intensity in a droplet of a quenched FAM-labeled sample during fading over a period of 10 minutes (the numbers correspond to the minutes of bleaching). EXAMPLES

 In the following, the invention will be explained in more detail with reference to the following examples, without being limited to these.

 Example 1 :

Detailed Description of the Examination / Determination of Dyes Which Have Different Properties with Respect to Their Fluorescence Intensity Change or Luminosity Change After a Treatment:

 A reaction mixture is prepared containing the following components:

 2x Finnzymes DyNAmo Flash Probe qPCR Kit master mix (Biozym, Hessisch Oldendorf, Germany),

 - 300 nM Primers (Biomers, Ulm, Germany)

 - 200 nM probes (Biomers, Ulm, Germany)

 - DNA / RNAse free water (Invitrogen, Carlsbad, Germany)

 5'-GGCAATTGCGGCATGTTCTTCC-3 '(Forward Primer E. coli),

5'-TGTTGCATTTGCAGACGAGCCT-3 '(Reverse Primer E. coli),

 5'-Atto488-ATGCGAACGGCGGCAACGGCAACATGT-BHQ1-3 '(probe E. coli),

 5'-GCTAGCGAAACAAACGCTCAGCAA-3 '(Forward Primer β, subtilis),

 5'-ACTTCCACCCGAAGATGAAGTGCT-3 '(Reverse Primer β, subtilis),

 5'-FAM-AGCTGGACAACAAGGTCAATTCGGCA-BHQ1 -3 '(probe B. subtilis),

FAM and Atto488 fluoresce at very similar wavelengths (518 and 523 nm, respectively).

 Two reactions were prepared, one with ß. added subtilis DNA and the other added to E. coli DNA. Both reactions were exposed to PCR conditions and the fluorescence signal was measured. Both measurements were carried out in the same color channel (510 ± 5 nm). After the PCR, the samples were exposed to strong light and the fluorescence of the samples was measured at intervals of one minute each. The intensity at the time t = 0 (ie before the beginning of the irradiation by strong light) is used as initial value to all following

Normalize fluorescence measurements.

 The following formula can be used for the calculation:

Fluorescence intensity _{t = x}

fluorescence intensity As can be seen from FIG. 8, the fluorescence intensity decreases when irradiated by white light for both samples. However, the fluorescence intensity of the FAM labeled PCR probe decreases faster than the fluorescence intensity of Atto488 labeled PCR probe. Thus, the two probes can be distinguished on the basis of the dyes, although both measurements were carried out in the same color channel.

 The dye assay method disclosed in this example can be used to determine the fluorescence intensity change of a dye caused by the treatment between the measurements.

Example 2:

 Detailed description of the investigation / determination of two dyes, the

different properties with regard to their fluorescence intensity change or

Have luminance change, for the detection of two Zielseguenzen:

 1. A DNA sample containing an unknown target sequence (either A or B) is prepared for performing a DNA detection reaction (PCR) as follows.

 2. The necessary reagents for the biochemical detection reaction are added, including:

 a. PCR master mix

 b. DNA RNAse free water

 c. 2 primers for target sequence A (300 nM)

 d. 2 primers for target sequence B (300 nM)

 e. Probe for target sequence A labeled with FAM as fluorophore and BHQ1 as quencher (200 nM)

 f. Probe for target sequence B labeled with Atto488 as fluorophore and BHQ1 as

 Quencher (200 nM)

 3. The sample is amplified under standard PCR conditions.

4. The fluorescence intensity of the sample is measured in the green channel (area in which the

 Fluorescence maxima of FAM i / nc / Atto488 are) measured and recorded.

 5. By exponential amplification of the target sequence and associated

Cleavage of the probe releases fluorophore. 6. An exponential increase of the fluorescence can be observed, which subsequently changes into a plateau phase (the fluorescence remains constant). Until the end of the PCR, it is unclear which target sequence has been amplified.

 7. The last value of the fluorescence measurement during PCR is saved

 (Fluorescence value = R).

 8. The sample is irradiated for 6 minutes with intense white light. At this point, other changes may be made as mentioned above, e.g.

 Temperature changes.

 9. The fluorescence of the sample is measured (fluorescence value = S).

10. A quotient T = S / R is calculated.

 11. A standard value table is used around the quotient T a dye

 assign

 a) If the quotient is less than 0.5, the dye is identified as FAM. In the

 Detected sequence is target sequence A.

 b) If the quotient is greater than 0.5, the dye is identified as Atto488. In the

 Detected sequence is target sequence B.

 c) Optional: A "gray area" eg of T = 0.45-0.55 can be introduced If one obtains a quotient T in this range during the experiment, no assignment can be made, which increases the already high security continue, eg for particularly critical diagnostic decisions.

 If necessary, steps 8 and 9 can be repeated. For this purpose, an algorithm can be created or applied that different weights different points, so as to make the most reliable assignment of the measurement to a dye. If a standard value table is not available in step 1 1, one can be created in a reference experiment. Example 3:

 A reference experiment can be designed as follows:

 1. The necessary reagents for the biochemical detection reaction are mixed, including:

 a. PCR master mix

 b. DNA RNAse free water

c. 2 primers for target sequence A (300 nM) d. 2 primers for target sequence B (300 nM)

e. Probe for target sequence A labeled with FAM as fluorophore and BHQ1 as quencher (200 nM)

f. Probe for target sequence B labeled with Atto488 as fluorophore and BHQ1 as

 Quencher (200 nM)

The reaction mixture is divided into several subvolumes. Some of these subvolumes (A) are spiked with the target sequence A. Some of these subvolumes (B) are spiked with target B sequence. Some of these subvolumes (C) are used without destination sequence as no template control (NTC).

The sample is amplified under standard PCR conditions.

The fluorescence intensity of the sample is measured in the green channel (area in which the

Fluorescence maxima of FAM i / nc / Atto488 are) measured and recorded.

Due to the exponential amplification of the target sequence and associated cleavage of the probe, fluorophore is released.

An exponential increase in fluorescence can be observed, which subsequently changes into a plateau phase (the fluorescence remains constant). Until the end of the PCR, it is unclear which target sequence has been amplified.

The last value of the fluorescence measurement during the PCR is stored

(Fluorescence value = V).

The sample is irradiated for 6 minutes with intense white light. At this point, other changes may be made as mentioned above, e.g.

Temperature changes.

The fluorescence of the sample is measured (fluorescence value = W).

A quotient (X = W / V) is calculated. For all subvolumes A (XA), the quotients are averaged and a standard deviation calculated. For all subvolumes B, the quotients (XB) are averaged and a standard deviation calculated. For all subvolumes C, the PCR data are checked for a significant signal increase. If so, the experiment is repeated.

Steps 1.-10. are repeated (several times) to determine an interexperiment variability.

All quotients XA and XB are averaged and transferred to a standard value table. The standard deviations for XA and XB are used for the assignment indicate the confidence intervals corresponding to the unknown measured values in categories A and B.

 Example 4:

 Example of the temperature behavior of FAM & Atto488:

 The sample is processed as in Example 2. Instead Atto488 and FAM Atto647N and Cy5 are used (maxima at 669 and 670 nm). In step 7, the sample is measured at 25 ° C. In step 8, the sample is heated to 80 ° C. In step 9, the sample is measured at 80 ° C.

It can be read from the curve in FIG. 9 which quotients (T from step 10) match the presence of specific target sequences. This graphic can be used instead of a standard value table in step 1 1 or be converted into one before it for easier handling.

 Example 5:

 Detailed description of the investigation / determination of four dyes, the

different properties with regard to their fluorescence intensity change or

Luminance change, for the detection of four target sequences:

 1. A DNA sample containing one or more unknown target sequences (mecA, mecC, nuc, or PVL) is prepared for performing a DNA detection reaction (PCR) as follows.

 2. The necessary reagents for the biochemical detection reaction are added, including:

 a. PCR master mix

 b. DNA RNAse free water

 c. 2 primers for target sequence mecA (700 nM)

 d. 2 primers for target sequence mecC (500 nM)

 e. 2 primers for target sequence nuc (500 nM)

 f. 2 primers for target sequence Panton-Valentine leucocidin (PVL) (500 nM)

 G. Probe for target sequence mecA labeled with Atto647N as a fluorophore (200 nM) and ZEN / IABkFQ as a quencher

H. Probe for target sequence mecC labeled with Cy5 as fluorophore (20 nM) and BHQ1 as quencher Probe for target sequence nuc labeled with Atto488 as a fluorophore (200 nM) and BHQ2 as a quencher

 j. Probe for target sequence PVL labeled with FAM as a fluorophore (200 nM) and BHQ2 as a quencher

3. The sample is amplified under standard PCR conditions.

 4. The fluorescence intensity of the sample is measured in the green channel (area in which the

 Fluorescence maxima of FAM i / nc / Atto488 are) measured and recorded.

 5. The fluorescence intensity of the sample is determined in the red color channel (area in which the

 Fluorescence maxima of Atto647N and Cy5 are) measured and recorded.

6. By exponential amplification of the target sequence and associated

 Cleavage of the probe releases fluorophore.

 7. An exponential increase in fluorescence can be observed below

 Course goes into a plateau phase (the fluorescence remains constant).

 8. The last value of the fluorescence measurement during PCR is saved.

9. The sample is irradiated for 1 minute with intense white light of an LED (20 W 230 V)

10. The fluorescence intensity of the sample is measured in the green and red channels

1 1. Steps 8 - 10 are repeated 6 times, so that a decay curve of the

 Fluorescence intensity, as shown in Figure 10, is determined for the sample.

 12. The characteristic decay curve determines which target sequence the sample contains

 Example 6:

 Reference experiments to determine the level of confidence as a function of the concentrations of the target sequences

 For the reference experiment, samples are prepared as outlined in Example 5, PCR is performed, and fluorescence is measured in the red and green color channels. To evaluate with which confidence level the 4-plex-PCR experiment targets in

can determine different concentrations, experiments were performed for both color channels with no, low, medium and high concentration of each target sequence and any combination. Figure 12 summarizes the combination of concentrations as well as the results of these experiments. These results show that the confidence levels for the mecA / mecC system are higher than for the nuc / PVL system.

Example 7: Monochrome multiplex in digital PCR (dPCR)

 Digital PCR is a biochemical method for determining the concentration of individual DNA sequences and, in contrast to PCR, uses isolation of the DNA molecules by limiting dilution and microfluidics in a large number of separate ones. In the droplet digital PCR (ddPCR), the reaction spaces are formed by drops.

 For a monochrome multiplexing in ddPCR drops were generated using a Bio-Rad QX100 system. These drops contain the PCR reaction mixture, primer (200 nM) and probes (300 nM) and DNA RNAse-free water analogously to Example 2. As DNA target sequence ß. subtilis DNA and E. coli DNA related. The probes for the target sequences contained Atto488 and FAM as fluorophores. In the drops, the samples were under standard PCR

Conditions amplified. Subsequently, the drops were transferred to a readout chip to determine the fluorescence with the aid of a LaVision Bioanalyzer. For the

Fluorescence measurement was a mercury vapor lamp with an excitation filter of 482 nm and an emission filter of 536 is used. Between fluorescence measurements, the samples were bleached with the scanner light of the LaVision Bioanalyzer for 1 minute. The

 Fluorescence intensity of the samples was measured once per minute to determine a decay curve. Fig. 14 shows characteristic decay curves of drops containing samples with Atto488, FAM or Atto488 and FAM. Fig. 5 shows fluorescence images of droplets containing FAM quenched hydrolysis samples and Atto488 quenched hydrolysis samples.

Example 8:

 Verification experiment with different concentrations of E. coli DNA and a constant B. subtilis DNA concentration

For a verification experiment different concentrations of E. coli DNA (220, 440, 880, 1760 cp. ΜΓ ¹ ) with a constant concentration of ß. subitlis DNA (200 cp. μΓ ¹ ) mixed. The DNA target sequence was mixed with a master mix comprising primers and probes for both target sequences and a droplet digital PCR (ddPCR) was performed therewith. For each concentration, the fluorescence intensity of 150 drops before and after fading was determined. The fluorescence measurement was performed every minute for a total bleaching time of 10 minutes. The intensity values at time 0 min were used to distinguish between positive and negative drops as a function of a limit value.

Subsequently, the intensity values of each drop were normalized to the value at time 0 min and set to 1. Thereafter, normalized fluorescence at 10 min was used to determine in which droplet the Atto488 probe was depleted (> 75% of the initial intensity) and in which droplet the FAM probe was de-quenched (<75% of the initial intensity). Using the Poisson distribution, the number of initial target DNA copies was determined from the number of positive amplified drops. With help bionomial statistics, the 95% confidence interval was determined. The results of the experiments are shown in Fig. 13A.

 Example 9:

 Verification experiment with different concentrations of B. subtilis DNA and a constant E. coli DNA concentration

The verification experiment according to Example 8 was repeated, with the difference that different concentrations of ß. subtilis DNA (200, 632, 2000 cp. μΓ ¹ ) in a constant concentration of E. coli DNA (600 cp. μΓ ¹ ) were determined. The results of these experiments are shown in Fig. 13B.

Claims

A method for distinguishing between the light emission and / or the light reflection of at least two dyes based on their emission and / or

 Reflection wavelength are indistinguishable, comprising the steps:

 a) providing the at least two dyes which are indistinguishable on the basis of their emission and / or reflection wavelength and are preferably present as a mixture in a sample;

 b) treatment of the at least two dyes, wherein the treatment causes a change in the light emission and / or the light reflection of at least one of the dyes; and

 c) measuring the combined light emission and / or the light reflection of the at least two dyes, at least before and after the treatment in step b); characterized in that

 d) the at least two dyes are differently changed by step b) with respect to their light emission and / or light reflection, and

 e) calculating the concentration of at least one of the dyes by changing the measured light emission and / or light reflection between the treatments in step c).

 2. The method according to claim 1, characterized in that

 the dyes are fluorescent dyes.

 3. The method according to claim 1 or 2, characterized in that

 the treatment in step b) leads to a different reduction in the intensity of the light emission and / or the light reflection of the respective dyes.

 4. The method according to any one of the preceding claims, characterized in that

 the treatment in step 1 b) a light irradiation comprises a temperature change, a Red / Ox potential change, a change of an electromagnetic field, a pH change, a surface change and / or a pressure change.

 5. Method according to the preceding claim, characterized in that

the light irradiation takes place with a corresponding excitation wavelength.

6. The method according to any one of the preceding claims, characterized in that the light emission and / or the light reflection of the dyes correlated with the concentration of biological molecules.

 7. The method according to any one of the preceding claims, characterized in that

 the dyes are bound to biological molecules.

 8. The method according to claim 6 and / or 7, characterized in that

 the biological molecules are nucleic acids, antibodies and / or antigens.

 9. The method according to any one of the preceding claims, characterized in that at least one of the dyes is in spatial proximity to a quencher, so that the measured light emission and / or the light reflection of the at least two dyes results from the sum of the non-quenched portions of the dyes, wherein the efficiency of the quencher is changed by step b), and when two or more quenchers are in spatial proximity to the at least two dyes, the efficiency of the respective quenchers is changed differently by step b).

 10. The method according to any one of the preceding claims, characterized in that the change in the light emission and / or the light reflection by step b) is irreversible.

1 1. A method according to any one of the preceding claims, characterized in that

 the change in the light emission and / or the light reflection through step b) is reversible or partially reversible.

 12. The method according to any one of the preceding claims, characterized in that

 the treatment in step b) is a light irradiation and this results in a first of the at least two dyes to a greater reduction of light emission and / or light reflection, as in a second of the at least two dyes and the

Calculation of the concentration in step e) by comparing the quotient of the combined light emission measured in step c) and / or the light reflection of the at least two dyes after and before the light irradiation (L _to / L _before ) with one or more threshold values

a) in the case that the quotient L _to / L _before greater than a first threshold, a higher concentration of the second dye compared to the first dye is present, and

b) in the event that the quotient L _to / L _before less than a second threshold, a higher concentration of the first dye compared to the second dye is present.

13. The method according to the preceding claim, characterized in that the first and the second threshold are identical and the method a

 Threshold includes.

 14. Method according to the preceding claim, characterized in that

the first threshold is greater than the second threshold, and in the case that the quotient L _na c _h Lvor is greater than or equal to the second threshold and less than the first threshold, the concentration of the first colorant is not different from the concentration of the second colorant.

 15. Method according to the preceding claim, characterized in that

 the threshold is between 0.2 and 0.8.

 16. The method according to the preceding claims 12-15, characterized in that the light irradiation takes place over a period of 30 seconds to 30 minutes.

 17. Method according to the preceding claim, characterized in that

 the light irradiation takes place over a period of 3 minutes to 12 minutes.

 18. The method according to any one of the preceding claims 12-17, characterized in that the first dye is selected from a group comprising FAM, Cy5, TET, JOE.

19. The method according to any one of the preceding claims 12-18, characterized in that the second dye is selected from a group comprising Atto488, Atto647, Atto532, Atto647N

 20. Method according to the preceding claim, characterized in that

 the determination of the dyes is carried out in a "real-time" assay.

 21. Method according to the preceding claim, characterized in that

 the "real-time" assay is a quantitative PCR (qPCR) or a quantitative reverse transcription PCR (qRT-PCR).

 22. Method according to the preceding claims, characterized in that the

 Calculation of the concentration of at least one of the dyes in step e) by means of a computer-implemented method.

23. Application of the method according to one of the preceding claims for the determination of two or more nucleic acids.

24. Application of the method according to the preceding claim, characterized in that the two or more nucleic acids nucleic acid products of a

 Amplification reaction are.

 25. Application of the method according to the preceding claim, characterized in that the amplification reaction is a polymerase chain reaction (PCR).

 26. Application of the method according to one of the preceding claims for the determination of two or more protein targets.

 27. Application of the method according to the preceding claims, characterized in that the two or more protein targets are antibodies or antigens.

 28. Application of the method according to the preceding claims, characterized in that the application is carried out in an immune or cell assay.

 29. Use of the method according to the preceding claim for the determination of two or more nucleic acids, wherein the dyes are bound to different oligonucleotides and the oligonucleotides enable a sequence-specific binding to one or more target sequences.

30. Application of the method according to the preceding claim, characterized in that the dye-oligonucleotide combinations are preferably selected from the following group: TaqMan probes, molecular beacons, hybridization probes, RPA probes, mediator probes, FRET probes and / or Q _G probes.

 31. A system for distinguishing between the light emission and / or the light reflection of at least two dyes that are indistinguishable based on their emission and / or reflection wavelength, comprising:

 a) at least two dyes which are (i) based on their emission and / or

 (Ii) show different changes in their light emission and / or light reflection, which are caused by the treatment according to claim 1 b), and (iii) are present as a mixture in a sample;

b) means for treating the at least two dyes, wherein the treatment causes a change in the light emission and / or light reflection of at least one of the dyes, c) means for measuring the light emission and / or the light reflection of the at least two dyes

 32. System according to the preceding claim, characterized in that

 the means for measuring the light emission and / or the light reflection of the at least two dyes is a fluorescence and / or a colorimetric detector.

 33. System according to one of the preceding claims, characterized in that

 Means for irradiating the at least two dyes are present in the system.

 34. System according to the preceding claim, characterized in that

 the means for irradiating the at least two dyes is a light-emitting diode (LED).

35. System according to one of the preceding claims, characterized in that

 the system comprises means for performing a computer-implemented method for calculating the concentration of at least one of the dyes.

 36. System according to the preceding claim, characterized in that

 the calculation of the concentration of the at least one dye according to the calculation of the concentration of the at least one dye in a method according to claims 1-30 is carried out.