Systems and methods for detecting analytes from multiplex assays

Abstract

Systems and methods to enable analyte detection in a multiplex amplification process can include obtaining composite emission signal data associated with composite emission signals from at least a first probe type and a second probe type at a plurality of points in time during the amplification process, the first probe type includes a first marker configured to generate a first transmit signal, and the second probe type includes a second marker configured to generate a second transmit signal having spectrally similar characteristics to the first transmit signal, the first probe type and the second probe type differ in thermal and / or temporal properties; and determining transmit signal data associated with a transmit signal from a given one of the first probe type or the second probe type during the amplification process based at least in part on the composite transmit signal data.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 356,874, filed on June 29, 2022, entitled “Systems and Methods for Enabling Multiplexed Polymerase Chain Reaction Processes,” U.S. Provisional Application No. 63 / 356,863, filed on June 29, 2022, entitled “Compositions and Methods for Detecting Nucleic Acids Using Intra-Channel Multiplexing,” and U.S. Provisional Application No. 63 / 408,665, filed on September 21, 2022, entitled “Compositions and Methods for Detecting Nucleic Acids Using Intra-Channel Multiplexing,” the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] Aspects of the present disclosure relate to systems and methods for multiplexed analyte detection. In particular, the present disclosure relates to nucleic acid detection using multiplexed nucleic acid amplification assays (eg, polymerase chain reaction assays). Background Art

[0004] Nucleic acid detection assay is usually carried out by adding a sample suspected of comprising one or more target nucleic acids to the reaction mixture.The reaction mixture may include one or more detectable labels, each of which is designed to be associated with different target nucleic acids, and produces a signal corresponding to the amount of the target nucleic acid associated in the reaction mixture. In "single plex" assay, the reaction mixture includes a single detectable label designed to be associated with a single target nucleic acid. On the contrary, in "multiplex" assay, the reaction mixture includes a plurality of different detectable labels, each of which is usually designed to have specificity to different target nucleic acids.

[0005] Thus, multiplexed assays are able to detect multiple different targets in a single reaction mixture.In some applications, the detectable label is a fluorescent dye incorporated into a nucleic acid probe, primer, or some other nucleic acid molecule designed to specifically hybridize to a corresponding target nucleic acid with which it is designed to associate.

[0006] Challenges arise when implementing multiplexed systems and processes for determining the relative amounts of different target nucleic acids in a sample. Specifically, the use of detectable labels that have spectral similarity (producing identical or overlapping emission signals) can make it difficult to determine the respective contribution of each label individually, and therefore the contribution of the respective different target nucleic acids with which they are associated.

[0007] There is a need to provide more robust systems and methods for performing multiplexed nucleic acid detection assays, such as, for example, nucleic acid detection utilizing various polymerase chain reaction (PCR) assays, and for analyzing data associated with such assays. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Various objects, features, characteristics, and advantages of the present invention within the scope of the present disclosure will become apparent and more readily understood from the following description of various embodiments taken in conjunction with the accompanying drawings and the appended claims, all of which form a part of this specification. In the drawings, like reference numerals may be used to indicate corresponding or similar parts in the various figures, and the various elements depicted are not necessarily drawn to scale, wherein:

[0009] Figure 1 The emission spectra of various fluorescent dyes that can be used in nucleic acid detection assays are shown;

[0010] Figure 2A is a schematic overview of techniques for achieving detection of multiple target nucleic acids using detectable labels having spectral similarity according to various embodiments of the present disclosure;

[0011] Figure 2B is a diagram showing various embodiments according to the present disclosure Figure 2A A plot of the signal response over time for the technique outlined in ;

[0012] Figure 3A shows the activity of cleavable and non-cleavable probes during the annealing step, the extension step, and the denaturation step of thermal cycling according to embodiments of the present disclosure;

[0013] Figure 3B is a graph showing fluorescence signal response over time during thermal cycling of an amplification process utilizing Figure 3A cleavable probes and non-cleavable probes;

[0014] Figure 4 A flow chart depicting exemplary actions associated with implementing analyte detection in a multiplex amplification process according to embodiments of the present disclosure is shown;

[0015] Figure 5A and Figure 5BShown are the results of a duplex assay in which TaqMan probes and extendible fluorescent (EF) probes were designed to produce spectrally similar fluorescent signals ( Figure 5A ) or produce fluorescent signals with different spectral profiles (e.g., no significant overlap) ( Figure 5B );

[0016] Figure 5C Compare to how to use Figure 5A The EF-related fluorescence signal derived from the assay results is similar to that in Figure 5B The EF-related fluorescence signal is directly measured in the assay.

[0017] Figure 5D The fluorescence signals of TaqMan probe and extendable fluorescent probe (EF) in the extension stage and denaturation stage are shown;

[0018] Figure 6 shows the results of another 9-plex assay test according to an embodiment of the present disclosure, which includes 5 different detectable labels, wherein the corresponding TaqMan probes and EF probes share four detectable labels and one detectable label is associated only with the TaqMan probe;

[0019] Figure 7A shows the process of using a tailed primer specific for a nucleic acid target to form a template to which an EF probe can hybridize; and

[0020] Figure 7B Examples of primers that can be included in the reaction mixture include a tail forward primer, a reverse primer, and an EF probe to achieve Figure 7A process. DETAILED DESCRIPTION

[0021] In the context of nucleic acid probes and target nucleic acids, the term "specifically interacts" (and similar terms) indicates that the probe is designed to interact with the target to a greater extent than with non-target nucleic acids also present in the reaction mixture. For example, a specific interaction can include hybridization of the probe in whole or in part to the corresponding target. The hybridization between the probe and the target does not need to be 100%. For example, a functionally effective interaction can be achieved with a probe that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or up to 100% homology to its corresponding target.

[0022] As used herein, " detection channel " is a specified subset of the total range of possible values ​​of a detectable signal. For example, when the detectable signal is a fluorescent signal, a detection channel (e.g., a fluorescent channel or a dye channel) can represent a wavelength band of a specified size. A detection channel can, for example, have a band size of about 10nm-60nm, depending on the instrument sensitivity and / or the desired signal granularity. A detection channel can also include a discontinuous wavelength or wavelength range. Depending on the filter arrangement for measuring the detectable signal, a detection channel can be defined additionally or alternatively. Each different detection channel typically includes a specific filter arrangement to block non-channel emission. Therefore, as a functional definition, each detectable signal within a given filter arrangement can be considered to be within the same detection channel.

[0023] As used herein, separate fluorescent signals having "substantially the same fluorescence" provide fluorescent emissions within a similar wavelength band. For example, a first fluorescent signal and a second fluorescent signal having substantially the same fluorescence may have emission peaks that differ by no more than about 10 nm, or no more than about 8 nm, or no more than about 6 nm, or no more than about 4 nm, or no more than about 2 nm, or no more than about 1 nm, or may be substantially indistinguishable from one another based on the sensitivity of the detection instrument used to measure the fluorescent emissions. Additionally or alternatively, in applications where the fluorescent signals are measured using the same filter arrangement, the fluorescent signals may be considered to have "substantially the same fluorescence."

[0024] As used herein, "substantial signal" and / or a detectable signal with "substantial fluorescence" is a signal significantly higher than background (i.e., baseline) level, including a fluorescent signal significantly higher than fluorescence background / baseline level. This can be defined by a threshold value that separates background fluorescence from substantial fluorescence. The threshold value can vary according to specific test protocols and application requirements. In some embodiments (without passive reference), the threshold value is set to, for example, about 1,000 to about 30,000, or more generally about 2,000 to about 20,000, or about 3,000 to about 15,000, or about 4,000 to about 6,000, or in the range of endpoints defined by any two values ​​in the aforementioned values. In some embodiments (for example, using passive reference), the threshold value is set to, for example, a change (ΔRn) of the fluorescence signal with baseline detrending, which is about 0.01 to 0.5. In some embodiments, the threshold value is higher than the baseline level in some percentages, such as about 5% to about 10% higher than the baseline level.

[0025] The "background" or "baseline" level of signal during the amplification process (i.e., the background / baseline level of fluorescence) can be determined according to methods known to those skilled in the art. As a non-limiting example, the baseline level can be determined as the median signal of the amplification cycle before exponential amplification occurs. For example, exponential amplification can be determined when the signal change from one amplification cycle to the next exceeds a certain percentage indicating an exponential change.

[0026] As a corollary, a signal and / or fluorescence level that is not "substantial" according to the foregoing may be described herein as "negligible." Similarly, a probe is "substantially bound" to its target when the probe is significantly above background (e.g., above binding to non-targets) relative to probe binding. Optionally, at least 1%, 5%, 10%, 20%, 50%, or 80% of the probe or target is bound.

[0027] As used herein, a "cleavable" probe is one that is intended to be cleaved as a result of the specific interaction of the probe with its corresponding target, thereby resulting in the release of the corresponding label and an increase in the corresponding detectable signal.

[0028] As used herein, "non-cleavable" probes are probes with a label that is intended to be associated with the probe throughout the assay. In non-cleavable probes, the corresponding detectable signal changes according to the configuration of the probe, rather than changing according to the release of the label on the probe. Extendable fluorescent probes are examples of non-cleavable probes. For example, extendable fluorescent probes can include universal or hairpin extendable fluorescent probes, as described in various embodiments, or can have the structure of a non-hairpin sequence.

[0029] The terms "detectable signal" and "label signal" are used synonymously in this article. For example, a "first label signal" is a signal emitted by a first label of a first probe type, and a "second label signal" is a signal emitted by a second label of a second probe type. A "total signal" is the total measured signal within a specific detection channel at a given time point or measurement point. A plurality of different "detectable signals" / "label signals" may contribute to the same "total signal". For example, the total signal may include a signal generated by a first label of a first probe type and a signal generated by a second label of a second probe type. In this regard, in some cases, the "total signal" may be regarded as a "composite signal". In some embodiments, the signal is a fluorescent signal, and terms such as "first fluorescent signal", "second fluorescent signal" and "total fluorescent signal" can be used as specific examples of corresponding broader terms.

[0030] The term "spectral similarity" refers to the emission signals of detectable labels having the same spectral profile or substantially overlapping spectral profiles. Thus, different probe types carrying the same detectable label or different probe types carrying different detectable labels have substantial spectral overlap in the emission signals, and both probes can be considered as probes with spectral similarity. In some embodiments, detectable labels with spectral similarity can be detected in the same optical detection channel, but other techniques can also be used to detect the emission signals of such detectable labels. Reference to substantially overlapping spectra should be understood to mean spectral similarity. Although spectral similarity refers to the fact that emission signals exhibit the same or a certain degree of spectral signal and profile overlap, the term does not refer to the time course or time period over which such signals with corresponding profiles are emitted, such as during different stages of the amplification process.

[0031] The term "end point" as used in connection with cycles is a specified cycle at which the PCR process is assumed to be complete and / or a specified cycle at which a signal threshold value higher than a specified amount of background signal appears. In various embodiments, the end point cycle according to the present disclosure may be in the range of 20 to 45 cycles, such as 30 to 40 cycles. However, the number of cycles to the end point cycle may be different from these ranges and may be a specified cycle and / or a cycle associated with the position at which the emission (e.g., fluorescence) signal indicating that the amplified product has reached a predetermined level. An "end point signal" refers to an emission signal measured during the end point cycle.

[0032] The terms "determine," "calculate," and "estimate" are used synonymously herein. These terms are not intended to imply a precise level of measurement accuracy. Thus, where values ​​are "determined," "calculated," or "estimated" using the embodiments described herein, it should be understood that such values ​​may include some degree of inherent error due to factors such as instrument tolerances, rounding, chemical reaction variability, and other inherent measurement imperfections known and understood by those skilled in the art.

[0033] For any given element of a component of a described embodiment, any possible alternatives listed for that element or component can generally be used alone or in combination with one another, unless implicitly or explicitly stated otherwise.

[0034] In addition, unless otherwise indicated, the numbers used in the specification and claims to express quantity, composition, distance or other measurements should be understood as being optionally modified by the term "about" or its synonyms. When the terms "about", "approximately", "substantially" and the like are used in conjunction with a stated amount, value or condition, they may mean an amount, value or condition that deviates from the stated amount, value or condition by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1% or less than 0.01%. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted in accordance with the number of reported significant figures and by applying ordinary rounding techniques.

[0035] Any headings and sub-headings used herein are for organizational purposes only and are not meant to limit the scope of the description or the claims.

[0036] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" do not include plural referents unless the context clearly dictates otherwise. Thus, for example, an embodiment referring to a singular referent (e.g., "a widget") may also include two or more such referents.

[0037] It should also be understood that the embodiments described herein may also include properties and / or features (e.g., ingredients, components, members, elements, parts, and / or portions) described in one or more separate embodiments, and are not necessarily strictly limited to the features explicitly described for that particular embodiment. Thus, the various features of a given embodiment may be combined with and / or incorporated into other embodiments of the present disclosure. Therefore, the disclosure of certain features with reference to a specific embodiment of the present disclosure should not be construed as limiting the application or incorporation of the features into that specific embodiment. On the contrary, it should be understood that other embodiments may also include such features.

[0038] Overview of Multiplexing Using Spectrally Similar Detectably Labeled Probes

[0039] In various multiple nucleic acid detection assays, each detectable label is assigned to different targets.Then, the presence and / or amount of each target can be determined by, for example, measuring the signal emitted from the detectable label in a separate " detection channel ", these separate detection channels each correspond to the specific properties of the signal emitted. For example, in the context of fluorescent emission dyes as detectable labels, separate detection channels can correspond to the emission wavelength spectrum relevant to each dye. However, a certain amount of overlap can be present in the emission spectra of different dyes. The overlap (spectral similarity) increased in the emission spectrum increases the difficulty of resolving the fluorescent emission signal detected separately, and therefore increases the difficulty of detecting and / or quantifying the corresponding target.

[0040] Although the purpose of selecting multiple dyes is to minimize spectral overlap, the limited nature of emission spectra imposes practical restrictions on the number of individual dyes that can be combined in the same multiple assay. Therefore, at present, there are significant restrictions on the number of different targets that can be detected and / or measured in multiple assays. Therefore, there is a need for compositions and methods that can increase the "diversity" of detection assays. In addition, it may be desirable to use detectable labels with spectral similarity, such as using dyes that have some degree of overlap in emission spectra, and / or using the same dye for different target nucleic acids.

[0041] Various embodiments disclosed herein relate to systems and methods for implementing multiple nucleic acid detection assays that rely on a polymerase chain reaction (PCR) process by enabling determination of separate detectable signals that are each associated with a different assay target nucleic acid, but that have spectral similarity. For example, various embodiments allow for detection of spectrally similar detectable labels within the same detection channel (e.g., within a channel sensitive to emission within a defined spectral range (e.g., fluorescence emission)).

[0042] Figure 1 The emission spectra 150 of various fluorescent dyes 152 that can be used in nucleic acid detection assays are shown. As described above, a multiplexed assay can assign each dye as a label to a separate target nucleic acid, and then determine the presence and / or amount of each target by measuring the fluorescent signal in a separate detection channel, each of which corresponds to a different emission wavelength of the corresponding dye. As shown, there can be some (e.g., substantial) overlap in the emission spectra 150 of one or more dyes 152, such that those dyes are spectrally similar. For example, Figure 1 As shown, AF647 and Cy5 dyes have spectral similarity in that they have essentially the same emission spectra. Although multiplex dyes are typically selected with the goal of minimizing spectral overlap, the finite nature of emission spectra actually limits the number of individual dyes that can be combined in the same multiplex assay, and therefore acts as a practical limit on the number of different targets that can be detected and / or measured without increasing system complexity (such as increasing the number of individual detection channels and / or implementing other relatively complex deconvolution schemes).

[0043] Various embodiments described herein solve one or more of the aforementioned problems by utilizing multiple detectable signals to realize analyte detection in multiple amplification process, and these multiple detectable signals are each associated with different determination target analytes or target analyte groups, have spectral similarity in their emission spectra, and / or can use the same detection channel to detect.Multiple detectable signals can be resolved individually and analyzed independently, thereby allowing detection and / or quantification of each target.By allowing the use of the same one or more detectable labels (e.g., dye) with spectral similarity to determine multiple target analytes, disclosed embodiments can beneficially increase "diversity" (i.e., the quantity of target analytes that can be detected and quantified in multiple determinations), without relying on additional detectable labels (e.g., dye), detection channels and / or the problem of accompanying spectral overlap.For example, according to aspects of the present disclosure, public detection channels can be used to detect labels with spectral similarity but intended for different target analytes.Similarly, embodiments described herein can beneficially reduce the quantity of the separate detectable labels (e.g., dye) required in multiple determinations, without reducing the diversity of determination. In addition, various embodiments can allow the use of the same detectable label (e.g., a dye) as a label for different target nucleic acids in multiple assays, including using the same label for different target nucleic acids in multiple assays, including in the same cycle of a PCR reaction. In addition, various embodiments can allow the detection of the same dye in the same detection channel.

[0044] Figure 2A 1. The present invention provides a schematic overview of a technique for detecting multiple target nucleic acids using a detectable label with spectral similarity by providing different first and second probe types, changing reaction mixture conditions, and measuring the resulting total signal under each set of conditions. As shown, a first probe 202 is designed to specifically interact with a first target 206. The first probe 202 includes a first label 210 that can produce a first label signal 214. The second probe 204 is designed to specifically interact with a second target 208 that is different from the first target 206. The second probe 204 includes a second label 212 that can produce a second label signal 216.

[0045] In some embodiments, the first marker 210 and the second marker 212 are identical. For example, the first marker 210 and the second marker 212 may comprise the same fluorescent dye. In some embodiments, the first marker 210 and the second marker 212 may be different, but still be designed to produce an emission signal having substantially the same or a certain degree of overlapping spectral profiles (e.g., having spectral similarity). For example, the first marker 210 and the second marker 212 may comprise a dye that is chemically different but functions to emit a signal having a similar wavelength. In some embodiments, the first marker signal 214 and the second marker signal 216 are measured using the same detection channel (e.g., including a filter arrangement) in a detection instrument.

[0046] The first probe 202 and the second probe 204 can be provided in the same reaction mixture and allowed to specifically interact with any first target 206 and second target 208 in the reaction mixture, respectively. As shown, the reaction mixture is subjected to at least two different sets of reaction conditions. The first probe 202 is designed so that during both the first set of conditions 218 and the second set of conditions 220, the first label 210 produces a first label signal 214, the extent of which is proportional to the amount of the specific interaction between the first probe 202 and the first target 206. On the contrary, the second probe 204 is designed so that the second label 212 produces a second label signal 216 during the second set of conditions 220 rather than during the first set of conditions 218, the extent of which is proportional to the amount of the specific interaction between the second probe 204 and the second target 208. In other words, under the first set of conditions 218, the first label signal 214 increases due to the specific interaction of the first probe 202 with the first target 206, but the second label signal 216 is not emitted due to the specific interaction of the second probe 204 with the second target 208. Under the second set of conditions 220, the second marker signal 216 increases due to the specific interaction of the second probe 204 with the second target 208, while the first marker signal 214 also further increases or remains at a level at least somewhat relatively increased compared to the first set of conditions.

[0047] During the first set of conditions 218, the second marker 212 will not generate a "substantial signal" (e.g., fluorescence), and therefore the second marker signal 216 will not be substantially different from the background (i.e., baseline) level of the emission signal (e.g., fluorescence) in the reaction mixture. That is, although there may be a certain non-zero level of signal generated by the second marker 212 during the first set of conditions 218, the second marker signal 216 will generally remain below a threshold that separates background signal from meaningful signal. As described above, this threshold can vary depending on the specific test protocol and application requirements.

[0048] In at least some embodiments, when both first target 206 and second target 208 are present in the reaction mixture, second marker signal 216 will differ to a greater extent between first set of conditions 218 and second set of conditions 220 than first marker signal 214 will differ to a greater extent between first set of conditions 218 and second set of conditions 220. Thus, while first marker signal 214 may differ somewhat between first set of conditions 218 and second set of conditions 220, the difference will generally be less than the difference in second marker signal 216 between first set of conditions 218 and second set of conditions 220.

[0049] Various embodiments of the present disclosure utilize the differences in the manner in which the first marker signal 214 and the second marker signal 216 respond to different sets of conditions so that the detected first marker signal 214 and the second marker signal 216 can be resolved (separated) even if, for example, they are detected within the same detection channel (e.g., for a given filter arrangement that filters an emission spectrum defined in the channel). As shown, the total signal (or composite signal) is measured during the first set of conditions 218, and the total signal ("second total signal") is measured during the second set of conditions 220. Fluorescence signal data representing the first total signal is sometimes referred to herein as "first fluorescence signal data," and fluorescence signal data representing the second total signal is sometimes referred to herein as "second fluorescence signal data" or "composite fluorescence signal data." As used herein, first and second in this context are not necessarily used to represent a temporal order of detections or conditions, although such a temporal order may occur.

[0050] During first set of conditions 218, the total signal will be substantially equal to first marker signal 214. That is, the first total signal will be primarily composed of first marker signal 214, with negligible contribution from second marker signal 216. During second set of conditions 220, the total signal will include a combination of first marker signal 214 and second marker signal 216. Thus, first marker signal 214 and second marker signal 216 can be resolved separately based on the first total signal and the second total signal. For example, first marker signal 214 can be determined based on the first total signal, and second marker signal 216 can be resolved by subtracting the first total signal from the second total signal.

[0051] In some embodiments, first marker signal 214 is directly equal to the first total signal. In other embodiments, first marker signal 214 is determined as a function of the first total signal. In some embodiments, the function is a linear function (although nonlinear functions may be used in some implementations). For example, as described above, even when the amount of first target 206 does not change, first marker signal 214 may differ slightly between first set of conditions 218 and second set of conditions 220. In some applications, first marker signal 214 under second set of conditions 220 may better correspond to a standard curve that equates first marker signal 214 to the amount of first target 206. Therefore, estimating first marker signal 214 as a function of the first total signal, rather than as being directly equal to the first total signal, may make the calculated first marker signal 214 closer to the signal that would be measured under second set of conditions 220 (i.e., second marker signal 216 without any interference).

[0052] In some embodiments, the function for converting the first total signal into the first label signal 214 is determined by comparing the first label signal 214 under the first set of conditions 218 with the first label signal 214 under the second set of conditions 220 in the absence of any second probe interacting with the second target. The first label signal 214 under the first set of conditions 218 and the second set of conditions 220 can be correlated with each other according to a linear function. In other embodiments, it can be correlated using a nonlinear function. When a linear function is used, a multiplication factor (e.g., a correction factor) can be used to convert the first total signal into the first label signal 214. Once such a linear function is determined, it can be used in subsequent determinations without the need for additional comparisons of the first label signal 214 under the first set of conditions 218 and under the second set of conditions 220 in the absence of the second probe having the second target. As described above, in some embodiments, the function for converting the first total signal into the first label signal can be nonlinear. In some embodiments, the function / correlation is determined between thermal cycles in which the number of expected cleavage probes is the same. For example, even if different signals are detected within the same detection channel, this method can also be used to resolve the different signals.

[0053] As described in more detail below, the first probe 202 and the second probe 204 have different mechanisms of action that achieve different signal responses to the first set of conditions 218 and the second set of conditions 220 based on the probe type. Beneficially, the ability to resolve the individual signals associated with each different probe type can be independent of the properties other than the different melting temperatures of the probes. Therefore, although the first probe 202 and the second probe 204 can have different melting temperatures, such different melting temperatures are not a prerequisite for allowing their associated marker signals to be effectively resolved. In some embodiments, for example, the melting temperature (Tm ) and T of the second probe 204 m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other, but such melting temperature differences do not limit the scope of the present disclosure. In addition, given the techniques for achieving differentiation of signal responses according to aspects of the present disclosure, there is no need to rely on the melting phase of the amplification process.

[0054] Figure 2B is a schematic diagram illustrating a method for reacting a first target 206 and a second target 208 based on cycling the reaction mixture between a first set of reaction conditions 218 and a second set of reaction conditions 220 and based on having both the first target 206 and the second target 208 present in the reaction mixture. Figure 2A . A cycle of conditions may include, for example, different conditions for various stages associated with thermal cycling in a nucleic acid amplification reaction (such as, for example, PCR). In such a reaction, a first set of reaction conditions 218 corresponds to a denaturation stage that supports thermal cycling, and a second set of reaction conditions 220 corresponds to an annealing and / or extension stage ("annealing / extension stage") that supports thermal cycling. Thus, in various embodiments, the first set of reaction conditions 218 includes a first temperature or temperature range, and the second set of reaction conditions includes a second temperature or temperature range (e.g., lower than the first temperature).

[0055] As shown in the figure, under second group of reaction conditions 220, first label signal 214 and second label signal 216 all increase.Under first group of reaction conditions 218, first label signal 214 keeps being roughly the same (although as mentioned above, it may change slightly) when previous cycle ends, and second label signal 216 drops to the level similar to the baseline signal level of second label signal 216, and this baseline signal level can be substantially constant in multiple amplification cycles.In other words, during first group of reaction conditions, second label signal 216 shows the baseline signal higher than background signal level.In some cases, second label signal can show the baseline signal level that changes at the different stages of amplification cycle, but still be enough to distinguish and be lower than the level under this second group of reaction conditions with the level under second group of reaction conditions.This may be due to the different states of probe and the proximity of quencher and mark.

[0056] As shown, first marker signal 214 and second marker signal 216 increase cumulatively with each successive occurrence of second set of conditions 220. This is a result of additional specific interactions in the reaction mixture between first probe 202 and first target 206, as well as additional specific interactions in the reaction mixture between second probe 204 and second target 208. However, as first marker signal 214 moves from the end of one cycle to the beginning of another (i.e., from the second set of conditions 220 at the end of a cycle to the first set of conditions 218 at the beginning of the subsequent cycle), second marker signal 216 returns to a level close to baseline at the beginning of each cycle (i.e., at each occurrence of first set of conditions 218). In other words, first marker signal 214 is continuous and accumulates in subsequent cycles, while second signal is transient and depends on the set of conditions occurring during a cycle.

[0057] Although some embodiments described herein can be used together with multiplexing in channel (using same detection channel detection mark), present disclosure is not limited thereto.In addition, present disclosure has envisioned that multiplexing in channel is combined with multiplexing between channels, to further increase the diversity of mensuration.For example, can design the mensuration with multiple different detectable labels (for example, dyestuff), these multiple different detectable labels have the spectrum for detecting using multiple different detection channels, wherein one or more channels in different channels are configured to use spectrally similar and can detect multiple detectable signals of the signal response from different targets according to the detectable signal that technology as described herein is resolved.In addition, by utilizing the various aspects of the probe with the detectable label that is designed to interact and show different transient emission signal patterns, it is possible to use additional probe types together with above-mentioned probe types, and measure in the additional time period of reaction cycle, so that it is possible to further distinguish which signals correspond to which target nucleic acids.

[0058] Cleavable and non-cleavable probes

[0059] In some embodiments, the first probe (e.g., first probe 202) is a "cleavable" probe. The first probe can be designed so that as a result of hybridization of the first probe to the first target (e.g., first target 206), the first label (e.g., first label 210) is separated from the first probe (and, for example, released from the corresponding quencher). Thus, once released, the first label continues to contribute to the total signal in the reaction mixture, thereby generating a cumulative emission signal. The first probe can be a TaqMan probe, for example, which undergoes cleavage during extension of the target molecule to which the probe hybridizes due to the 5' to 3' exonuclease activity of a DNA polymerase. TaqMan probes are described in U.S. Patent Nos. 4,889,818, 5,079,352, 5,210,015, 5,436,134, 5,487,972, 5,658,751, 5,210,015, 5,487,972, 5,538,848, 5,618,711, 5,677,152, 5,723,591, 5,773,258, 5,789,224, 5,834,836, and 7,445,900, all of which are hereby incorporated by reference herein.

[0060] In some embodiments, the second probe (e.g., second probe 204) is a "non-cleavable" probe. The label of the non-cleavable probe is intended to remain associated with the probe throughout the assay, and the level of the signal generated changes according to the probe configuration rather than the release of the label. The second probe can be an extendible fluorescent probe (EF), for example, which quenches the label when in a single-stranded configuration, but allows signal (i.e., generates a transient emission signal) when incorporated into a double-stranded molecule.

[0061] The EF probe can be, for example, a universal extendable fluorescent probe, an extendable hairpin probe designed for specific target amplification, or an extendable probe having a non-hairpin sequence structure.

[0062] Figure 3AShown during the annealing phase, extension phase and the denaturation phase of the PCR reaction thermal cycle, the activity of cleavable probe 302 and non-cleavable probe 312, this cleavable probe can be a TaqMan probe in various embodiments, and this non-cleavable probe can be an EF probe in various embodiments.As shown in the figure, during the annealing phase, TaqMan probe 302 and its corresponding target nucleic acid amplifier 304 (as used herein, target nucleic acid amplifier can refer to the single strand of target double-stranded nucleic acid, and should be understood by the context when describing the PCR reaction with reference to) hybridization.During the extension period of the primer 303 hybridized with the target nucleic acid amplifier 304 in probe 302 upstreams, 5 ' to 3 ' exonuclease activity of archaeal dna polymerase is cracked from the remainder of probe 302, thereby it is separated with corresponding TaqMan probe quencher 309.This causes the corresponding increase of fluorescent signal.During denaturation, mark 306 keeps free in the reaction mixture solution, and therefore continues to contribute to total fluorescent signal.

[0063] EF 312 comprises an EF label 316 and an EF quencher 319, which remain in close proximity when the extendable probe 312 is in a single-stranded configuration. Thus, when the EF is in a single-stranded configuration, the fluorescence signal from label 316 remains substantially quenched. During the annealing and extension phases, EF 312 hybridizes with its corresponding target template amplicon 314 and extends to form an extended probe amplicon 313. The extension of target template 314 then forms a complement 315 of the extended probe amplicon 313. The resulting double-stranded amplicon 317 forces label 316 away from quencher 319, to a distance sufficient to allow fluorescence emission. During denaturation, the extended probe amplicon 313 separates from its complement 315. Upon returning to a single-stranded configuration, label 316 and quencher 319 are brought back into proximity, and fluorescence is once again quenched.

[0064] Figure 3B 30 is a graph showing the fluorescence signal from the TaqMan probe 302 and the EF probe 312 over time during the thermal cycling of the amplification process. The temperature of the thermal cycle can be varied as needed for a particular application. For example, the denaturation phase can be performed at a temperature in the range of about 80°C to about 100°C, such as about 85°C to about 95°C, or such as about 90°C to about 95°C. The annealing / extension phase can be performed at a lower temperature, such as in the range of about 40°C to about 75°C, such as about 50°C to about 70°C, such as about 55°C to about 65°C. In some embodiments, the first set of reaction conditions (e.g., reference Figure 2A The first set of conditions discussed 218) corresponds to the denaturation stage 318, while the second set of reaction conditions (e.g., Figure 2A The second set of conditions discussed (220) corresponds to the annealing / extension phase 320.

[0065] In some embodiments, the amplification process is circulated between two target temperatures, for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycles of the amplification process.

[0066] Figure 3B As shown, the fluorescent signal associated with the TaqMan probe 302 increases during the extension phase 320 and then remains at a similar level during the denaturation phase 318 of the next cycle (although some relatively insignificant decrease in signal may occur as described above), while the fluorescent signal associated with the EF probe 304 increases during the extension phase 320, but then decreases to the baseline signal level associated with the EF probe 304 once the subsequent denaturation phase 318 reaches the target denaturation temperature. As will be appreciated by those of ordinary skill in the art, Figure 3B The cycles N, N+1, N+2 may start at different stages, however, in which case the above-mentioned comparison of the signal levels may be shifted.

[0067] In some embodiments, the first set of reaction conditions (e.g., denaturation conditions 318) includes a first measurement temperature at which the first marker signal is measured, and the second set of reaction conditions (e.g., annealing / extension conditions 320) includes a second, different measurement temperature at which the first marker signal and the second marker signal are measured. In some embodiments, the first measurement temperature and the second measurement temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more. The first measurement temperature can be, for example, a target denaturation temperature in the range of about 80°C to about 100°C, for example, about 85°C to about 95°C, or for example, about 90°C to about 95°C, and the second measurement temperature can be, for example, a target annealing / extension temperature in the range of about 40°C to about 75°C, for example, about 50°C to about 70°C, for example, about 55°C to about 65°C.

[0068] Example Techniques and Implementations

[0069] The disclosed techniques for achieving analyte detection in a multiplex amplification process may require performing various actions. For example, Figure 4 An example flow chart 400 is shown that depicts actions associated with implementing analyte detection in a multiplex amplification process (eg, a multiplex polymerase chain reaction (PCR) process).

[0070] Action 402 of flowchart 400 comprises obtaining emission (e.g., fluorescence) signal data associated with a composite emission (e.g., fluorescence) signal from at least a first probe type including a first detectable label (e.g., fluorophore) and a second probe type at one or more time points during one or more cycles of an amplification process, the first probe type including a first detectable label (e.g., fluorophore) and the second probe type including a second detectable label (e.g., fluorophore) having spectral characteristics that are spectrally similar to the first detectable label. The first probe type and the second probe type may be selected / configured such that their associated label signals respond differently to different sets of reaction conditions (e.g., similar to those described above with reference to Figure 2A The first probe 202 and the second probe 204 discussed above). For example, the first probe type and the second probe type may differ in thermal and / or temporal properties. As described above, the first detectable label and the second detectable label may have spectral similarities such that both can be configured to produce a signal having a substantially similar wavelength range (e.g., corresponding to the wavelengths described above with reference to FIG. Figure 2A The emission signal (e.g., fluorescence) of the first and second labels discussed 212).

[0071] The composite signal may include an emission (e.g., a fluorescent signal) to which both the first probe type and the second probe type substantially contribute. As described above, emission (e.g., fluorescent) signal data associated with such a composite signal (and captured during specific reaction conditions, such as specific temperature conditions) may be considered "composite emission signal data" or "second emission signal data" or "composite fluorescent signal data" or "second fluorescent signal data."

[0072] As will be discussed in more detail below, Figure 5A An example illustration of composite fluorescent signal data from a first probe type and a second probe type is provided (e.g., where a TaqMan probe and an EF probe are labeled with a FAM fluorophore, the FAM signal at 64° C. (top row) represents the composite fluorescent signal). In some cases, the first fluorophore of the first probe type and the second fluorophore of the second probe type are the same (e.g., in Figure 5A, where both the TaqMan probe and the EF probe are FAM-labeled). Despite the potential commonality between the fluorophores of the two probe types, the two probe types can be configured in other ways to have corresponding binding affinities for different analytes (e.g., different target nucleic acids). For example, a first probe type can have a binding affinity for a first analyte (e.g., corresponding to the first probe 202 being designed to interact with the first target 206, as described above with reference to Figure 2A ), and the second probe type may have a binding affinity for a second analyte different from the first analyte (e.g., corresponding to the second probe 204 being designed to interact with the second target 208, as discussed above with reference to Figure 2A discussed).

[0073] In some embodiments, one probe type (e.g., a first probe type) is a cleavable probe, and the other probe type (e.g., a second probe type) is non-cleavable. In some embodiments, the cleavable probe type is a TaqMan probe, and the non-cleavable probe type is an EF probe (e.g., in reference Figure 3A 、 Figure 3B and 5A to 5D In the examples discussed above, the first probe type can be associated with a cumulative fluorescence that remains substantially stable over multiple segments of an amplification cycle and increases cumulatively over multiple amplification cycles, while the second probe type can be associated with a transient fluorescence that fluctuates significantly with each amplification cycle, such as during different stages of the amplification cycle.

[0074] Action 404 of flowchart 400 includes determining, during one or more cycles of the amplification process, emission (e.g., fluorescence) signal data associated with the emission (e.g., fluorescence) signal from a given probe type based at least in part on the emission (e.g., fluorescence) signal data associated with the composite emission signal and thermal and / or temporal properties of the first probe type and / or the second probe type. The given probe type associated with the determined emission (e.g., fluorescence) signal may include at least one of the first probe type or the second probe type (e.g., Figure 5C derived EF-related signals).

[0075] In some implementations, the first emission signal data is acquired at multiple time points during the amplification process from which the composite emission signal data is obtained. The first emission signal data can be associated with reaction conditions that are different from the reaction conditions associated with the composite emission signal data. For example, the composite emission signal data can be associated with a second set of reaction conditions (e.g., corresponding to the reaction conditions described above). Figure 2A 、 Figure 2B and Figure 3BThe second set of conditions 220, 320 discussed above) are associated with the first emission signal data, and the first emission signal data can be associated with the first set of reaction conditions (e.g., corresponding to the first set of reaction conditions discussed above). Figure 2A 、 Figure 2B and Figure 3B The first set of conditions discussed are associated with 218, 318).

[0076] In some cases, different sets of reaction conditions are associated with different temperature conditions, such as different temperatures or different temperature ranges (e.g., a first temperature condition associated with a first emission signal data, and a second temperature condition associated with a second emission signal data or a composite emission signal data). In some cases, the first temperature condition is higher than the second temperature condition and / or does not overlap with the second temperature condition. For example, Figure 3B A second temperature condition of 65° C. is depicted, which may be associated with composite emission signal data, and a first temperature condition of 95° C., which may be associated with second emission signal data. Other temperature conditions are within the scope of the present disclosure (e.g., a second temperature condition in the range of approximately 40° C.-75° C.; a first temperature condition in the range of approximately 80° C.-100° C.). The first temperature condition and the second temperature condition may be associated with different stages of the amplification process. For example, the second temperature condition may be associated with the annealing stage or the extension stage or a combined extension / annealing stage of the amplification process (e.g., Figure 3B ), while the first temperature condition may be associated with the denaturation phase of the amplification process (e.g., Figure 3B A first set of conditions 318 for the various amplification cycles represented in FIG.

[0077] In view of the reaction differences in the reaction conditions, the composite emission signal data and the first emission signal data can be associated with different time points or time periods during the amplification process. For example, the data points (or subsets of data points) that form the composite emission signal and the first emission signal data can be acquired / captured in a temporally staggered manner (e.g., alternating between capturing data points for the composite emission signal data and capturing data points for the first emission signal data as the reaction conditions alternate during the amplification process). In addition, the first emission signal and the composite emission signal can be captured by collecting several data points during the amplification (e.g., PCR) cycle and separating the two signals according to their different temporal characteristics / profiles. As described above, by utilizing the different temporal aspects of the various emission signals, the technology can also be used for more than two probe types with detectable labels.

[0078] Determining the shot data associated with a shot of a given probe type may utilize the composite shot data (discussed above with reference to action 402) and the first shot data as input, as will be discussed below with reference to Figure 5A A more detailed description (e.g., briefly refer to Figure 5A, the signal data in the top row, left column, represents example measured composite fluorescence signal data; the signal data in the middle row, left column, represents example transformed first fluorescence signal data determined using the measured first fluorescence signal data; and the signal data in the bottom row, left column, represents example fluorescence signal data associated with the fluorescence signal of a given probe. Fluorescence signal data associated with the fluorescence signal of a given probe can be determined in real time during the amplification process, or as a post-processing operation.

[0079] Figure 5A and Figure 5B Results are shown for a qPCR duplex assay where TaqMan and EF probes were designed to produce signals that are spectrally similar and detectable, for example, in the same dye channel ( Figure 5A ) or produce spectra that do not have significant overlap and are detectable, for example, in different dye channels ( Figure 5B ) different fluorescence signals. Figure 5A In the assay shown, both the TaqMan probe and the EF probe were labeled with FAM, and thus the VIC fluorophore signal (in Figure 5A The right-hand column of the data results is depicted in Figure 2) as a control. Figure 5B In the assay shown, the TaqMan probe was labeled with VIC and EF was labeled with FAM. The reaction mixture composition, template DNA concentration, and amplification conditions remained unchanged in both assays.

[0080] exist Figure 5A , graph 502 shows in the left column the change in FAM fluorescence signal (ΔRn) (y-axis) as a function of cycle number (x-axis) measured at the temperature during the annealing / extension phase (64° C. in this example) with the baseline signal detrended. This signal is expected to include fluorescence generated by both TaqMan probe labels (those that have been cleaved from the probe) and EF probe labels (those that have been incorporated into the double-stranded amplicon). Figure 5A The FAM signal of FIG502 may include composite fluorescence signal data, as described above with reference to Figure 4 discussed.

[0081] Figure 5A Graph 504 shows the baseline detrended variation (ΔRn) of the FAM fluorescence signal as a function of cycle number measured at the temperature during the denaturation phase (95°C in this example) and modified by a linear function that relates the 95°C measurement to the 64°C measurement of the TaqMan probe. The signal indicates the approximate fluorescence associated with the TaqMan probe at 95°C and is expected to include fluorescence resulting from the TaqMan probe label but not significant fluorescence from the EF probe label. The signal is measured at 95°C and linearly transformed to provide Figure 5AThe FAM fluorescence signal data of the signal data shown in Figure 504 may include the above reference Figure 4 The "first fluorescence signal data". Therefore, Figure 5A The transformed fluorescence signal data depicted in graph 504 of can be considered "transformed first fluorescence data." As described above, the function used to relate the 95°C measurement to the 64°C measurement can be determined by comparing TaqMan probe signals measured under different temperature conditions (e.g., in the absence of other probe types that are similar or spectrally similar to the EF probe, e.g., in the same spectral channel).

[0082] exist Figure 5A In the example of FIG. 1 , composite fluorescence signal data (e.g., at a second set of reaction conditions, i.e., at an annealing or extension stage temperature (or a combined annealing / extension stage of 64° C.)) is measured. Figure 5A 502 ), and capture fluorescence signals from two probe types (i.e., TaqMan probes and EF probes). According to a first set of reaction conditions (i.e., at a denaturation stage temperature of 95° C.), (e.g., Figure 5A 504 ) of FIG transforming the first fluorescent signal data to approximate the fluorescent signal from one of the probe types (i.e., the TaqMan probe type). As described above, the composite fluorescent signal data and the transformed first fluorescent signal data can be used to determine the fluorescent signal from the other probe type (the EF probe type) according to the second set of reaction conditions (i.e., at an annealing or extension temperature of 64° C.). The composite fluorescent signal data can be modified by the transformed first fluorescent signal data (e.g., by subtracting the transformed first fluorescent signal data from the composite fluorescent signal data) to generate fluorescent signal data for the EF probe type (e.g., as discussed herein as a “given probe type”).

[0083] Figure 5A Graph 506 shows a resolved fluorescence signal determined by subtracting the signal of graph 504 (i.e., the transformed first fluorescence signal) from the signal of graph 502 (i.e., the composite fluorescence signal). This signal is intended to be used to estimate the fluorescence generated by the EF label, separate from the fluorescence generated by the TaqMan probe label. Therefore, this signal can be used to quantify / analyze targets (e.g., one or more target nucleic acids) associated with the EF in the reaction mixture. The first fluorescence data or the transformed fluorescence signal data can be used to quantify / analyze different targets associated with the TaqMan probe in the reaction mixture.

[0084] Thus, the disclosed technology can advantageously enable the assaying of multiple targets / analytes within the same reaction mixture using probes with detectable labels (e.g., fluorophores) that are spectrally similar and, for example, detectable within the same detection channel (e.g., within the same fluorescence channel) through the same amplification process, thereby beneficially enabling increased diversity in multiplexed assays without reliance on additional dyes.

[0085] Figure 5B The graph representation of Figure 5A Same signal measurement type, but in the generated Figure 5B In the determination of the data of , the EF probe is labeled with FAM dye, and the TaqMan probe is labeled with VIC dye. Figure 508 shows the fluorescence signal generated by the EF label (FAM), and Figure 510 shows the fluorescence signal generated by the TaqMan probe label (VIC). Figure 512 shows the insignificant change in the fluorescence generated by the EF label at the denaturation temperature (that is, the EF label fluorescence is basically maintained at its baseline signal level), while Figure 514 shows the fluorescence signal generated by the TaqMan probe label, which is basically equal to (after applying the linear function) the signal measured at the annealing / extension temperature. Because the TaqMan probe and the EF probe are differentially labeled in this determination, Figure 516 shows the resolved signal of the EF label, which basically matches the EF signal (in Figure 508) at the annealing / extension temperature, and Figure 518 shows the expected baseline signal obtained by subtracting the TaqMan label signal during the denaturation period from the TaqMan label signal during the annealing / extension period of the TaqMan probe. The EF-related fluorescence signal of Figure 516 therefore closely represents a direct measurement of the signal from the EF label (e.g., of Figure 508).

[0086] Figure 5C Compared with Figure 5A The determination of the derived EF-related fluorescence signal 520 ( Figure 5A 506) and the measured EF-related fluorescence signal 522 ( Figure 5B Figure 516). The results show that the resolved signal and the measured signal are closely related. Therefore, the results show that the fluorescence signals generated by different probe types using the same label in the same detection channel can be resolved separately.

[0087] For further reference, Figure 5D Shown are the fluorescence signals as a function of cycle number measured with the TaqMan probe and EF probe combination during the annealing / extension phase (in this example at 65°C) and during the denaturation phase (in this example at 95°C).

[0088] Figure 6Shown is the result of another determination test comprising 5 different detection channels / dyes, wherein four have TaqMan probes and EF probes, have the same label (dyes) in each channel and have different labels between the four channels. One channel has only TaqMan probes in the channel. The result shows that the fluorescent signals of different probe types can be determined independently, and therefore 9-plex reactions can be effectively performed.

[0089] In view of the foregoing, in some embodiments, the disclosed embodiments may include obtaining different emission (e.g., fluorescence) signals under different temperatures, time periods (e.g., reaction phases of thermal cycling), or other conditions of an amplification process (e.g., PCR process) involving multiple different probe types. Different probe types may include different emission signal (e.g., fluorescence) responses at different temperatures or time periods. Different emission signal responses of different probe types under different temperatures, time periods, or other conditions may be used to determine isolated emission signals of different probe types. Thus, this can achieve multiplex PCR assays, which can quantify different target analytes using emission signals that emit the same or substantially overlapping emission spectra (spectrally similar), and thus, for example, a common detection channel can be utilized to detect multiple analytes.

[0090] End-point PCR implementation plan

[0091] The analysis techniques according to various embodiments may also be used when performing a traditional endpoint PCR process, wherein the sample is subjected to batch PCR (or a larger reaction volume, not intended to capture single or no DNA molecules using Poisson statistics), and as will be familiar to those of ordinary skill in the art. In such a PCR process, the measurement of the signals from the two different probe types may occur at the end cycle of the PCR and under different reaction conditions (such as, for example, denaturation conditions and annealing and / or extension conditions as described herein), similar to the methods described above. In the case of an endpoint PCR process, the detected marker signal will follow Figure 2A The signals outlined in , wherein the signals are thus indicative of the presence or absence of the respective first and second targets. Figure 3B , the measured endpoint cycle signals under two different reaction conditions can therefore result in different signal levels schematically shown at cycle N+2 (analogously to the endpoint cycle). By comparing the different signal levels obtained, using Figure 2A The algorithms set forth in and further described above can determine the presence or absence of a first target nucleic acid and a second target nucleic acid using detectable labels that are spectrally similar and detectable in the same detection channel.

[0092] Thus, methods for determining the presence or absence of multiple targets using the multiplexing techniques described herein can also be performed using endpoint PCR (i.e., using a whole or larger reaction volume whose size is not intended to rely on Poisson statistics and capturing a single molecule of target nucleic acid in the reaction volume, as is familiar to those of ordinary skill in the art). Methods employed in endpoint PCR applications include: preparing a reaction mixture comprising a first probe type (e.g., a TaqMan probe) and a second probe type (e.g., an EF probe), the first probe type and the second probe type being configured to specifically interact with a first nucleic acid target and a second nucleic acid target, respectively; subjecting the reaction mixture to an amplification reaction (e.g., PCR); measuring an endpoint cycle signal of the reaction mixture under a first set of reaction conditions (e.g., denaturing conditions, such as about 95°C); and measuring an endpoint signal of the reaction volume under a second set of reaction conditions (e.g., annealing / treatment conditions, such as about 65°C); The method further comprises measuring a first total emission (e.g., fluorescence) signal including any first emission (e.g., fluorescence) signal (if present) during the reaction conditions, measuring a second total emission (e.g., fluorescence) signal including any first emission (e.g., fluorescence) signal (if present) and any second emission (e.g., fluorescence) signal (if present) during the second set of reaction conditions, and estimating the first emission (e.g., fluorescence) signal and / or the second emission (e.g., fluorescence) signal based on the first total emission (e.g., fluorescence) signal and the second total emission (e.g., fluorescence) signal to determine the presence or absence of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture. In other words, the above reference to Figure 5A and Figure 5B The described techniques can be applied to end-point PCR assays and analytical procedures.

[0093] Stretchable fluorescent (EF) probe template formation

[0094] Figure 7A The process of forming a target template 1014 to which an EF probe 1012 can hybridize is shown using a tailed primer 1022 that is specific for a nucleic acid target 1024. The tailed primer 1022 includes a tail portion 1026 and a target-specific portion 1028. Figure 7B An example of a more detailed view of a tailed primer 1022 as a forward primer, a target-specific primer 1023 paired with the tailed primer 1022 as a reverse primer, and the EF probe 1012 is shown.

[0095] As shown, in the first stage, target-specific portion 1028 hybridizes to target 1024. Extension of target-specific portion 1028 forms tailed amplicon 1025. Primer 1023, paired with tailed primer 1022, enables extension of the complement of tailed amplicon 1025. It is this complement that forms target template 1014. As shown, target template 1014 includes tail complement portion 1027.

[0096] In the second stage, the EF probe 1012 is hybridized to the target template 1014, and amplification can continue, e.g., as Figure 3A As shown, EF probe 1012 includes a probe tail 1017 that has substantial homology to tail 1026 and is therefore complementary to tail complement portion 1027 of target template 1014. Extension of probe 1012 and target template 1014 forms a double-stranded amplicon 1019. Primer 1023, shown here paired with tailed primer 122, can also be used as primer 1023 paired with EF probe 11012 to enable formation of double-stranded amplicon 1019, as shown. Figure 3A shown.

[0097] like Figure 7B As shown, tail 1026 can be formed with the 5' end of tail primer 1022. EF probe 1012 can include a stem-loop portion, wherein stem portion 1010 is on either side of loop portion 1011, configured to form a stem-loop structure when EF probe 1012 is single-stranded. For example, label 1016 can be located on one side of the stem-loop portion, and quencher 1018 can be located on an opposite side of the stem-loop portion, such that when the stem-loop structure is formed, label 1016 and quencher 1018 are in close proximity, but when EF probe 1012 is constrained to a more linear configuration (e.g., when incorporating a double-stranded amplicon), the label and quencher are spaced further apart.

[0098] In the embodiment shown, the label 1016 is located at or near the 5' end of the EF probe 1012, and the quencher 1018 is located at the 3' end of the label 1016. In other embodiments, the positions of the label 1016 and the quencher 1018 can be reversed. Preferably, as shown, the stem-loop portion is disposed at the 5' end of the probe tail 1017, so that the stem-loop portion remains at the end of the amplicon generated by extension of the EF probe 1012, thereby making it less likely that the stem-loop structure formation (when single-stranded) will be compromised.

[0099] In addition to or as an alternative to the EF probes described herein, some embodiments may include other labeled oligonucleotides that produce increased fluorescence when incorporated into double-stranded amplicons (relative to when in a single-stranded state), such as, for example, during the extension and / or annealing phases of a PCR process. For example, LUX TMThe primer contains an internal fluorophore that is quenched by a hairpin structure located 5' of the fluorophore. Like the EF probe, when incorporated into the double-stranded amplicon, the LUX TM The primers provide increased fluorescence, and the hairpin structure is linearized. Additionally, any primer or probe described herein may include one or more locked nucleic acids (LNAs) known in the art.

[0100] In some embodiments, tailed primer 1022 and corresponding (tailless) primer 1023 are provided at different concentrations. For example, primer 1023 can be provided at a higher concentration than tailed primer 1022. For example, primer 1023 can be provided at a concentration of about 2X (2 times) to about 30X the concentration of tailed primer 1022, or about 5X to about 25X the concentration of tailed primer 1022, or about 10X to about 20X the concentration of tailed primer 1022. Because primer 1023 can play a role in (1) driving the formation of target template 1014 (e.g., Figure 7A ) and (2) driving the formation of complement 1015 of the extended probe amplicon 1013 (as shown Figure 3A ), so providing this primer at a higher concentration than the corresponding tailed primer 1022 can beneficially balance the reaction and help drive overall reaction efficiency.

[0101] In some embodiments, EF probe 1012 is provided at a concentration that is different from the concentration of tailed primer 1022 and / or the concentration of primer 1023. For example, EF probe 1012 can be provided at a concentration that is greater than the concentration of tailed primer 1022 and less than the concentration of primer 1023. In some embodiments, EF probe 1012 is provided at a concentration that is about 2X to about 20X the concentration of tailed primer 1022, or about 3X to about 15X the concentration of tailed primer 1022. As described above, providing a relatively high concentration of primer 1023 helps drive the overall efficiency of the reaction. Compared to tailed primer 1022, providing EF probe 1012 at a higher concentration than tailed primer 1022, but not necessarily higher than primer 1023, drives more of the associated amplification toward EF probe 1012, while still allowing primer 1023 to be the primary driver of reaction efficiency.

[0102] In addition to or as an alternative to using a "universal" EF probe with a probe tail 1017, other embodiments include and / or utilize EF probes having a target-specific portion instead of a probe tail 1017. Such EF probes can hybridize directly to a target template nucleic acid, such as Figure 7A , for generating a target template 114 having a tail complement portion 1027. In such an embodiment, the probe tail 1017 of the EF probe 1012 is replaced with a target-specific portion that hybridizes directly to the target 1024. The process is otherwise similar to Figure 3AThat is, after extension of the EF probe, a subsequent round of annealing / extension will extend the complementary strands to form a double-stranded amplicon that separates the fluorophore and quencher to allow for the generation of a fluorescent signal.

[0103] Additional Marker / Dye Details

[0104] Exemplary, non-limiting detectable labels that can be used in the embodiments described herein include, for example:

[0105] Fluorescein (e.g., 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfluorescein (6-FAM), VIC, FITC, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE)), 5- and 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 5- and 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514));

[0106] Pyrenes; (e.g., Cascade Blue; Alexa Fluor 405);

[0107] Coumarins (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430);

[0108] Cyanine dyes (e.g., Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7);

[0109] Rhodamines; (e.g., 110, 123, B, B 200, BB, BG, B extra, 5 and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5 and 6-carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5 and 6-ROX (carboxy-X-rhodamine), Sulforhodamine B can C, Sulforhodamine G Extra, 5 and 6TAMRA (6-carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X);

[0110] Alexa Fluor fluorophores (a broad class that includes many dye types, such as cyanines) (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 676, 680, 700, 750);

[0111] FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine); and other types of dyes known to those skilled in the art.

[0112] Fluorophore labels can be associated with quenchers such as dark fluorescence quenchers (DFQ), black hole quenchers (BHQ), Iowa Black, QSY7, QSY21 quenchers, Dabsyl and Dabcel sulfonate / carboxylate quenchers, and MGB-NFQ quenchers. For example, fluorophore labels can also include sulfonate derivatives of fluorescein dyes with SO3 replacing the carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0113] Additional Amplification Details

[0114] The amplification products produced using one or more embodiments described herein can be produced, detected and / or analyzed on any suitable platform. In some embodiments, the nucleic acid target can be single-stranded, double-stranded or any other nucleic acid molecule of any size or conformation. Amplification process as described herein can include PCR (see, e.g., U.S. Patent number 4,683,202). In various embodiments, the PCR process can be real-time or quantitative PCR (qPCR), endpoint PCR, digital PCR (dPCR) or any combination thereof.

[0115] In some embodiments, the amplification process includes reverse transcription PCR (RT-PCR). The disclosed methods may include, for example, subjecting the target nucleic acid to a reverse transcription reaction prior to amplification by PCR. In some embodiments, the amplification process includes a one-step RT-PCR (e.g., in a single container or reaction volume) in which one or more reverse transcriptases are used in combination with one or more DNA polymerases.

[0116] Optionally, certain qPCR assays can be plated in individual wells of a single array or multi-well plate, such as a TaqMan Array Card (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346800 and 4342265) or a MicroAmp multi-well (e.g., 96-well, 384-well) reaction plate (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346906, 4366932, 4306737, 4326659, and N8010560). Optionally, the different qPCR assays present in different wells of an array or plate can be dried or freeze-dried in situ, and the array or plate can be stored or shipped before use. In some embodiments, the concepts described herein can be used in in situ hybridization applications that are not necessarily associated with PCR.

[0117] Other amplification methods, such as, for example, loop-mediated isothermal amplification ("LAMP") and other isothermal methods can also be used with the assay embodiments described herein.

[0118] The components described herein for multiplexing using spectrally similar detectably labeled probes can be provided in a kit together with one or more additional components to facilitate the amplification process. Such components can include, for example, dNTPs, DNA polymerase, amplification buffers / reagents, master mix components known in the art, and other components known in the art for achieving or assisting nucleic acid amplification.

[0119] Additional implementation details

[0120] The principles disclosed herein can be implemented in various formats. For example, the various techniques discussed herein can be implemented as various actions (e.g., Figure 4 In addition to the various techniques described above for collecting data at multiple time points during each cycle of the amplification process (e.g., qPCR assay), data can also be collected at the endpoint cycle of the amplification process (e.g., endpoint PCR assay) and analyzed according to the techniques described herein. This endpoint data collection technique can be used, for example, in genotyping applications. The various techniques disclosed for data collection and analysis are not mutually exclusive and can be used in combination. In some cases, the techniques discussed herein are represented by computer-executable instructions that can be stored on one or more hardware storage devices. The computer-executable instructions may be executed by one or more processors to implement (or configure the system to implement) the disclosed techniques. In some embodiments, the system may be configured to send computer-executable instructions to a remote device to configure the remote device to perform the disclosed techniques.

[0121] The various specific temperatures, times, and temperature change rates depicted in the figures and discussed with respect to the various embodiments are merely illustrative and should not be construed as limiting the scope of the present disclosure and the claims. Other specific temperatures, times, and temperature change rates may be employed and will be understood from the remainder of this disclosure. In addition, the different sets of conditions under which the detection of the emission signal occurs may differ from one another in different cycles. For example, the denaturation temperature may vary from cycle to cycle, but is still considered to be a common set of conditions for performing the measurements. Similarly, various annealing phase and / or extension phase conditions may be employed in different cycles.

[0122] Systems for implementing the disclosed embodiments may include various components such as, by way of non-limiting example, processors, memory, sensors, I / O systems, communication systems, and the like.

[0123] A processor may include one or more sets of electronic circuits containing any number of logic units, registers, and / or control units to facilitate the execution of computer-readable instructions (e.g., instructions forming a computer program). Such computer-readable instructions may be stored in a storage device. The storage device may include physical system memory and may be volatile, non-volatile, or some combination thereof. Furthermore, the storage device may include a local storage device, a remote storage device (e.g., accessible via a communication system or other means), or some combination thereof.

[0124] In some implementations, the processor may include or may be configured to execute any combination of software components and / or hardware components operable to facilitate processing using a machine learning model or other artificial intelligence-based structure / architecture. For example, the processor may include and / or utilize hardware components or computer-executable instructions to execute functional blocks and / or processing layers configured in the form of, as non-limiting examples, a single-layer neural network, a feedforward neural network, a radial basis function network, a deep feedforward network, a recurrent neural network, a long short-term memory (LSTM) network, a gated recurrent unit, an autoencoder neural network, a variational autoencoder, a denoising autoencoder, a sparse autoencoder, a Markov chain, a Hopfield neural network, a Boltzmann machine network, a restricted Boltzmann machine network, a deep belief network, a deep convolutional network (or convolutional neural network), a deconvolutional neural network, a deep convolutional inverse graph network, a generative inverse network, a liquid machine, an extreme learning machine, an echo state network, a deep residual network, a Kohonen network, a support vector machine, a neural Turing machine, and / or others.

[0125] In some cases, actions that may be performed by the system may rely, at least in part, on a communication system for receiving data from a remote system, which may include, for example, a separate system or computing device, a sensor, and / or the like. The communication system may include any combination of software or hardware components operable to facilitate communication between components / devices on the system and / or with components / devices external to the system. For example, the communication system may include a port, bus, or other physical connection device for communicating with other devices / components. Additionally or alternatively, the communication system may include systems / components operable to wirelessly communicate with external systems and / or devices via any suitable communication channel, such as, by way of non-limiting example, Bluetooth, ultra-wideband, WLAN, infrared communication, and / or the like.

[0126] The system may include or communicate with a sensor. A sensor may include any device for capturing or measuring data representing a perceptible or detectable phenomenon. As non-limiting examples, a sensor may include one or more light sensors / detectors, microphones, thermometers, barometers, magnetometers, accelerometers, gyroscopes, and / or the like.

[0127] In addition, the system may include or communicate with an I / O system. The I / O system may include any type of input or output device, such as, by way of non-limiting example, a touch screen, a mouse, a keyboard, a controller, a speaker, and / or the like, but not limited thereto. For example, the I / O system may include a display system, which may include any number of display panels, optical devices, laser scanning display assemblies, and / or other components. In view of this disclosure, it should be understood that in some cases, sensors may be used as an I / O system.

[0128] The disclosed embodiments may include or utilize a special-purpose or general-purpose computer including computer hardware, as discussed in more detail below. The disclosed embodiments also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media can be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer-readable media that store computer-executable instructions in the form of data are one or more "physical computer storage media" or "hardware storage devices." Computer-readable media that only carry computer-executable instructions without storing computer-executable instructions are "transmission media." Thus, by way of example and not limitation, the present embodiments may include at least two distinct types of computer-readable media: computer storage media and transmission media.

[0129] Computer storage media (also called "hardware storage devices") are computer-readable hardware storage devices based on RAM, flash memory, phase change memory ("PCM"), or other types of memory, such as RAM, ROM, EEPROM, CD-ROM, solid-state drives ("SSD"), or other optical disk storage, magnetic disk storage, or other magnetic storage devices, or any other medium that can be used to store the desired program code tools in the form of computer-executable instructions, data, or data structures in hardware and that can be accessed by a general-purpose or special-purpose computer.

[0130] A "network" may include one or more data links that enable the transmission of electronic data between computer systems and / or modules and / or other electronic devices. When information is transferred or provided to a computer over a network or other communications connection (hardwired, wireless, or a combination of hardwired or wireless), the computer properly views the connection as a transmission medium. Transmission media may include networks and / or data links that can be used to carry program code in the form of computer-executable instructions or data structures and that can be accessed by general-purpose or special-purpose computers. Combinations of the foregoing are also included within the scope of computer-readable media.

[0131] Furthermore, program code tools in the form of computer-executable instructions or data structures can be automatically transferred from a transmission computer-readable medium to a physical computer-readable storage medium (and from a physical computer-readable storage medium to a transmission computer-readable medium) upon reaching various computer system components. For example, computer-executable instructions or data structures received over a network or data link can be cached in RAM within a network interface module (e.g., a "NIC") and then ultimately transferred to computer system RAM and / or a less volatile computer-readable physical storage medium at the computer system. Thus, a computer-readable physical storage medium can be included in a computer system component that also (or even primarily) utilizes a transmission medium.

[0132] Computer executable instructions include, for example, instructions and data that cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions. Computer executable instructions may be, for example, binary, intermediate format instructions (such as assembly language), or even source code.

[0133] The disclosed embodiments may include or utilize cloud computing. Cloud models may include various characteristics (e.g., on-demand self-service, broad network access, resource pooling, rapid elasticity, measured services, etc.), service models (e.g., software as a service ("SaaS"), platform as a service ("PaaS"), infrastructure as a service ("IaaS")), and deployment models (e.g., private cloud, community cloud, public cloud, hybrid cloud, etc.).

[0134] Those skilled in the art will appreciate that the present invention can be practiced in a network computing environment with many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile phones, PDAs, pagers, routers, switches, wearable devices, and the like. The present invention can also be implemented in a distributed system environment, where multiple computer systems (e.g., local and remote systems) linked by a network (by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) perform tasks. In a distributed system environment, program modules can be located in local and / or remote memory storage devices.

[0135] Alternatively or additionally, the functions described herein may be performed at least in part by one or more hardware logic components. For example, but not limited to, illustrative types of hardware logic components that may be used include field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), application specific standard products (ASSPs), systems on chips (SOCs), complex programmable logic devices (CPLDs), central processing units (CPUs), graphics processing units (GPUs), and / or other components.

[0136] As used herein, the terms "executable module," "executable component," "component," "module," or "engine" may refer to a hardware processing unit or a software object, routine, or method that can be executed on one or more computer systems. The various components, modules, engines, and services described herein may be implemented as objects or processors (e.g., as separate threads) that execute on one or more computer systems.

[0137] In various embodiments, a system for detecting an analyte in a multiplex polymerase chain reaction (PCR) process includes one or more processors of at least one computing device; and a memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to perform the following process: obtaining measured emission signal data associated with a composite emission signal from at least a first probe type and a second probe type at one or more time points during one or more cycles of the PCR process, the first probe type may include a first label configured to produce a first emission signal, the second probe type may include a second label configured to produce a second emission signal, the second emission signal having spectrally similar characteristics to the first emission signal, the first probe type and the second probe type differing in thermal and / or temporal properties; and determining resolved emission signal data associated with a corresponding emission signal from one or more of the first probe type and the second probe type during the one or more cycles of the PCR process based, at least in part, on the emission signal data associated with the composite emission signal and the thermal and / or temporal properties of one or more of the first probe type and the second probe type.

[0138] Additional specific implementations of the system may also include any combination of one or more of the following features. The first label is identical to the second label. The first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, respectively. The acquisition occurs during a first set of reaction conditions and a second set of reaction conditions of an endpoint cycle of the PCR process, wherein the first set of reaction conditions and the second set of reaction conditions are different from each other. The amplification process is an endpoint PCR process. The first probe type has a binding affinity for a first analyte, and the second probe type has a binding affinity for a second analyte that is different from the first analyte. The determination may be performed in real time during the one or more cycles of the PCR process. The acquisition and determination may be performed by different processors. The first probe type is cleavable, and the second probe type is non-cleavable. The first probe type may include a Taqman probe, and the second probe type may include an extendable probe. The non-cleavable probe type may include an extendable fluorescent probe. The instructions, when executed by the one or more processors, further cause the one or more processors to perform the following process: obtaining additional measured emission signal data associated with a first temperature condition and / or time period of the one or more cycles during the PCR process, and wherein the measured emission signal data associated with the composite emission signal is associated with a second temperature condition and / or time period of the one or more cycles, the second temperature condition and / or time period being different from the first temperature condition and / or time period. The determining may utilize the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as inputs for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type. Utilizing the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as inputs for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type may include: generating transformed emission signal data by applying a transformation to the additional emission signal data; and modifying the measured emission signal data associated with the composite emission signal using the transformed emission signal data to generate the resolved emission signal data associated with the corresponding emission signal from the given probe type. The transformed emission signal data may indicate approximate emissions associated with the first probe type at the second temperature condition and / or time period.

[0139] The one or more instructions of the system, when executed by the one or more processors, may further cause the one or more processors to perform the following process: quantifying a first target associated with the first probe type based at least on the additional measured emission signal data; and quantifying a second target associated with the second probe type based at least on the resolved emission signal data associated with the corresponding emission signal from the given probe type. Quantifying the first target may include determining the concentration of the first target in the sample subjected to the PCR process, and quantifying the second target may include determining the concentration of the second target in the sample subjected to the PCR process. The first temperature condition and / or time period do not overlap with the second temperature condition and / or time period. The first temperature condition is higher than the second temperature condition. The second temperature condition is within a range of approximately 45°C to approximately 75°C. The first temperature condition is within a range of approximately 80°C to approximately 100°C. The second temperature and / or time condition is associated with an annealing phase, an extension phase, or a combined annealing and extension phase of the PCR process. The first temperature condition and / or time period is associated with a denaturation phase of the PCR process. The PCR process may include multiple PCR cycles. The first probe type can be associated with a cumulative emission signal that remains substantially stable across multiple stages of a PCR cycle and increases cumulatively across multiple PCR cycles. The second probe type can be associated with a transient emission signal that fluctuates across multiple stages in each PCR cycle. Specific implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0140] In various embodiments, a method for implementing analyte detection in a multiplex polymerase chain reaction (PCR) process may include: obtaining measured emission signal data associated with a composite emission signal from at least a first probe type and a second probe type at one or more time points during one or more cycles of the PCR process, the first probe type may include a first marker configured to emit a first emission signal, the second probe type may include a second marker configured to emit a second emission signal, the second emission signal having spectrally similar characteristics to the first emission signal, the first probe type and the second probe type differing in thermal properties and / or temporal properties; and determining, during the one or more cycles of the PCR process, resolved emission signal data associated with a corresponding emission signal from a given probe type of the first probe type and the second probe type based, at least in part, on the emission signal data associated with the composite emission signal and the thermal properties and / or temporal properties of one or more of the first probe type and the second probe type.

[0141] Additional specific implementations of this method may also include any combination of one or more of the following features. The first label may be the same as the second label. The first label and the second label may be a first fluorophore and a second fluorophore, respectively, and the first emission signal and the second emission signal may be a first fluorescent signal and a second fluorescent signal, respectively. The acquisition may occur during a first set of reaction conditions and a second set of reaction conditions of the endpoint cycle of the PCR process, wherein the first set of reaction conditions and the second set of reaction conditions are different from each other. The amplification process may be an endpoint PCR process. The first probe type may have a binding affinity for a first analyte, and the second probe type may have a binding affinity for a second analyte that is different from the first analyte. The first probe type may be cleavable, and the second probe type may be non-cleavable. The first probe type may include a Taqman probe, and the second probe type may include an extendable fluorescent probe. The non-cleavable probe type may be an extendable fluorescent probe.

[0142] The method may also include obtaining additional measured emission signal data associated with a first temperature condition and / or time period of the one or more cycles of the PCR process, and wherein the measured emission signal data associated with the composite emission signal is associated with a second temperature condition and / or time period of the one or more cycles, the second temperature condition and / or time period being different from the first temperature condition and / or time period. The determining may utilize the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as inputs for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type. Utilizing the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as inputs for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type may include: generating transformed emission signal data by applying a transformation to the additional emission signal data; and modifying the measured emission signal data associated with the composite emission signal using the transformed emission signal data to generate the resolved emission signal data associated with the corresponding emission signal from the given probe type. The transformed emission signal data may indicate approximate emissions associated with the first probe type at the second temperature condition and / or time period.

[0143] The method may also include any of the actions described as executable by the above instructions.

[0144] In other implementations, the computer-readable medium may store one or more instructions that may further implement and / or perform the actions of the method implementation using one or more of the combinations of features as recited above for the system.

[0145] It will also be understood how any feature or operation disclosed herein can be combined with any one or combination of other features and operations disclosed herein. In addition, the content or features in any one figure can be used in combination with any content or features used in any other figure. In this regard, the content disclosed in any one figure is not mutually exclusive and can be combined with content from any other figure.

[0146] The described embodiments are to be considered in all respects only as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the claims are intended to be embraced within their scope.

Claims

1. A system for detecting an analyte in a multiplex polymerase chain reaction (PCR) process, the system comprising: one or more processors of at least one computing device; as well as A memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to perform the following process: obtaining measured emission signal data associated with a composite emission signal from at least a first probe type and a second probe type at one or more time points during one or more cycles of the PCR process, the first probe type comprising a first label configured to produce a first emission signal, the second probe type comprising a second label configured to produce a second emission signal, the second emission signal having spectrally similar properties to the first emission signal, the first probe type and the second probe type differing in thermal and / or temporal properties; as well as During the one or more cycles of the PCR process, resolved emission signal data associated with a corresponding emission signal of a given probe type from one or more of the first probe type and the second probe type is determined based at least in part on the emission signal data associated with the composite emission signal and thermal and / or temporal properties of one or more of the first probe type and the second probe type.

2. The system of claim 0, wherein the first marker is the same as the second marker.

3. The system of claim 0 or claim 0, wherein the first probe type has a binding affinity for a first analyte and the second probe type has a binding affinity for a second analyte different from the first analyte.

4. The system of any one of claims 0 to 10, wherein the determining is performed in real time during the one or more cycles of the PCR process.

5. The system according to any one of claims 1 to 3, wherein the obtaining and determining are performed by different processors.

6. The system of any one of claims 0 to 5, wherein the first probe type is cleavable and the second probe type is non-cleavable.

7. The system of claim 6, wherein the first probe type comprises a TaqMan probe and the second probe type comprises an extendable probe.

8. The system of claim 6, wherein the non-cleavable probe type comprises an extendable fluorescent probe.

9. The system according to any one of claims 0 to 8, wherein the instructions, when executed by the one or more processors, further cause the one or more processors to perform the following process: obtaining additional measured emission signal data associated with first temperature conditions and / or time periods of the one or more cycles during the one or more cycles of the PCR process, and Wherein the measured emission signal data associated with the composite emission signal is associated with a second temperature condition and / or time period of the one or more cycles, the second temperature condition and / or time period being different from the first temperature condition and / or time period.

10. The system of claim 9, wherein the determining utilizes the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type.

11. The system of claim 1, wherein utilizing the measured emission signal data and the additional measured emission signal data associated with the composite emission signal as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type comprises: generating transformed transmit signal data by applying a transform to the additional transmit signal data; as well as The measured emission signal data associated with the composite emission signal is modified with the transformed emission signal data to produce the resolved emission signal data associated with the corresponding emission signal from the given probe type.

12. The system of claim 11, wherein the transformed emission signal data indicates an approximate emission associated with the first probe type at the second temperature condition and / or time period.

13. The system of claim 11 or claim 12, wherein the one or more instructions, when executed by the one or more processors, further cause the one or more processors to perform the following process: quantifying a first target associated with the first probe type based at least on the additional measured emission signal data; and A second target associated with the second probe type is quantified based at least on the resolved emission signal data associated with the corresponding emission signal from the given probe type.

14. The system of claim 13, wherein quantifying the first target comprises determining a concentration of the first target in a sample subjected to the PCR process, and quantifying the second target comprises determining a concentration of the second target in a sample subjected to the PCR process.

15. The system of any one of claims 9 to 14, wherein the first temperature condition and / or time period does not overlap with the second temperature condition and / or time period.

16. The system of any one of claims 9 to 15, wherein the first temperature condition is higher than the second temperature condition.

17. The system of any one of claims 9 to 16, wherein the second temperature condition is in the range of about 45°C to about 75°C.

18. The system of any one of claims 9 to 17, wherein the first temperature condition is in the range of about 80°C to about 100°C.

19. The system of any one of claims 9 to 18, wherein the second temperature and / or time condition is associated with an annealing phase, an extension phase, or a combined annealing and extension phase of the PCR process.

20. The system of any one of claims 9 to 18, wherein the first temperature condition and / or time period is associated with a denaturation phase of the PCR process.

21. The system of any one of claims 9 to 20, wherein the PCR process comprises a plurality of PCR cycles.

22. The system of claim 21, wherein the first probe type is associated with a cumulative emission signal that remains substantially stable during multiple stages of PCR cycling and that increases cumulatively over multiple PCR cycles.

23. The system of any one of claims 21 and 22, wherein the second probe type is associated with a transient emission signal that fluctuates over multiple stages in each PCR cycle.

24. The system of any one of claims 1 and 2, wherein the first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, respectively.

25. The system of any one of claims 1 and 2, wherein the obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an endpoint cycle of the PCR process, wherein the first set of reaction conditions and the second set of reaction conditions are different from each other.

26. The system of any one of claims 1 and 2, wherein the amplification process is an endpoint PCR process.

27. A method for achieving analyte detection in a multiplex polymerase chain reaction (PCR) process, the method comprising: obtaining measured emission signal data associated with a composite emission signal from at least a first probe type and a second probe type at one or more time points during one or more cycles of the PCR process, the first probe type comprising a first label configured to emit a first emission signal, the second probe type comprising a second label configured to emit a second emission signal, the second emission signal having spectrally similar properties to the first emission signal, the first probe type and the second probe type differing in thermal and / or temporal properties; as well as During the one or more cycles of the PCR process, resolved emission signal data associated with a corresponding emission signal from a given probe type from the first probe type and the second probe type is determined based at least in part on the emission signal data associated with the composite emission signal and thermal and / or temporal properties of one or more of the first probe type and the second probe type.

28. The method of claim 27, wherein the first label is the same as the second label.

29. The method of claim 27 or claim 28, wherein the first probe type has a binding affinity for a first analyte and the second probe type has a binding affinity for a second analyte different from the first analyte.

30. The method of any one of claims 37 to 29, wherein the determining is performed in real time during the one or more cycles of the PCR process.

31. The method of any one of claims 27 to 30, wherein the first probe type is cleavable and the second probe type is non-cleavable.

32. The method of claim 31, wherein the first probe type comprises a TaqMan probe and the second probe type comprises an extendable fluorescent probe.

33. The method of claim 31, wherein the non-cleavable probe type is an extendable fluorescent probe.

34. A method according to any one of claims 35 to 33, comprising: obtaining additional measured emission signal data associated with first temperature conditions and / or time periods during one or more cycles of the PCR process, and Wherein the measured transmit signal data associated with the composite transmit signal is associated with a second temperature condition and / or time period, wherein the second temperature condition and / or time period is different from the first temperature condition and / or time period.

35. The method of claim 34, wherein the determining utilizes the measured emission signal data associated with the composite emission signal and the additional measured emission signal data as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type.

36. The method of claim 35, wherein utilizing the measured emission signal data and the additional measured emission signal data associated with the composite emission signal as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type comprises: generating transformed transmit signal data by applying a transform to the additional measured transmit signal data; as well as The measured emission signal data associated with the composite emission signal is modified with the transformed emission signal data to produce the resolved emission signal data associated with the corresponding emission signal from the given probe type.

37. The method of claim 36, wherein the transformed emission signal data indicates approximate emissions associated with the first probe type at the second temperature condition and / or time period.

38. The method of claim 36 or claim 37, further comprising: quantifying a first target associated with the first probe type based at least on the additional measured emission signal data; as well as A second target associated with the second probe type is quantified based at least on the resolved emission signal data associated with the corresponding emission signal from the given probe type.

39. The method of any one of claims 34 to 38, wherein the first temperature condition and / or time period does not overlap with the second temperature condition and / or time period.

40. The method of any one of claims 34 to 39, wherein the first temperature condition is higher than the second temperature condition.

41. The method of any one of claims 34 to 40, wherein the second temperature condition is in the range of about 45°C to about 75°C.

42. The method of any one of claims 34 to 41, wherein the first temperature condition is in the range of about 80°C to about 100°C.

43. The method of any one of claims 34 to 42, wherein the second temperature condition and / or time period is associated with an annealing phase or an extension phase of the PCR process.

44. A method according to any one of claims 34 to 043, wherein the first temperature and / or time period is associated with a denaturation phase of the PCR process.

45. The method of any one of claims 34 to 44, wherein the PCR process comprises a plurality of PCR cycles.

46. ​​The method of claim 45, wherein the first probe type is associated with a cumulative emission signal that remains substantially stable over multiple fractions of PCR cycles and that increases cumulatively over multiple PCR cycles.

47. The method of any one of claims 45 to 46, wherein the second probe type is associated with a transient emission signal that fluctuates over multiple stages in each PCR cycle.

48. The method of any one of claims 27 and 28, wherein the first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, respectively.

49. The method of any one of claims 27 and 28, wherein the obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an endpoint cycle of the PCR process, wherein the first set of reaction conditions and the second set of reaction conditions are different from each other.

50. The method according to any one of claims 27 and 28, wherein the amplification process is an endpoint PCR process.

51. A computer readable medium storing one or more instructions that, when executed by one or more processors of at least one computing device, cause the one or more processors to perform the following process: obtaining measured emission signal data associated with a composite emission signal from at least a first probe type and a second probe type at one or more time points during one or more cycles of a PCR process, the first probe type comprising a first label configured to produce a first emission signal, the second probe type comprising a second label configured to produce a second emission signal, the second emission signal having spectrally similar properties to the first emission signal, the first probe type and the second probe type differing in thermal and / or temporal properties; and During the one or more cycles of the PCR process, resolved emission signal data associated with a corresponding emission signal from a given probe type from at least the first probe type and the second probe type is determined based at least in part on the emission signal data associated with the composite emission signal and thermal and / or temporal properties of one or more of the at least the first probe type and the second probe type.

52. The computer-readable medium of claim 51, wherein the first tag is the same as the second tag and the first transmit signal is the same as the second transmit signal.

53. The computer readable medium of claim 51 or claim 52, wherein the first probe type has a binding affinity for a first analyte and the second probe type has a binding affinity for a second analyte different from the first analyte.

54. The computer-readable medium of any one of claims 51 to 53, wherein the determining is performed in real time during the one or more cycles of the PCR process.

55. The computer readable medium of any one of claims 51 to 54, wherein the first probe type is cleavable and the second probe type is non-cleavable.

56. The computer-readable medium of claim 55, wherein the first probe type comprises a TaqMan probe and the non-cleavable probe type comprises an extendable fluorescent probe.

57. The computer readable medium of claim 55, wherein the non-cleavable probe type is an extendable fluorescent probe.

58. A computer readable medium according to any one of claims 51 to 57, The one or more instructions, when executed by the one or more processors, further cause the one or more processors to perform the following process: obtaining additional measured emission signal data associated with first temperature conditions and / or time periods during one or more cycles of the PCR process, and Wherein the measured transmit signal data associated with the composite transmit signal is associated with a second temperature condition and / or time period, wherein the second temperature condition and / or time period is different from the first temperature condition and / or time period.

59. The computer-readable medium of claim 58, wherein the determining utilizes the measured emission signal data and the additional measured emission signal data associated with the composite emission signal as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type.

60. The computer-readable medium of claim 59, wherein utilizing the measured emission signal data and the additional measured emission signal data associated with the composite emission signal as input for generating the resolved emission signal data associated with the corresponding emission signal from the given probe type comprises: generating transformed transmit signal data by applying a transform to the additional measured transmit signal data; as well as The measured emission signal data associated with the composite emission signal is modified with the transformed emission signal data to produce the resolved emission signal data associated with the corresponding emission signal from the given probe type.

61. The computer readable medium of claim 60, wherein the transformed emission signal data indicates an approximate emission associated with the first probe type at the second temperature condition and / or time period.

62. The computer readable medium of claim 60 or claim 61, wherein the one or more instructions, when executed by the one or more processors, further cause the one or more processors to perform the following process: quantifying a first target associated with the first probe type based at least on the additional measured emission signal data; and A second target associated with the second probe type is quantified based at least on the resolved emission signal data associated with the corresponding emission signal from the given probe type.

63. The computer readable medium of any one of claims 58 to 62, wherein the first temperature condition and / or time period does not overlap with the second temperature condition and / or time period.

64. The computer readable medium of any one of claims 58 to 63, wherein the first temperature condition is higher than the second temperature condition.

65. The computer readable medium of any one of claims 58 to 64, wherein the second temperature condition is in the range of about 45°C to about 75°C.

66. The computer readable medium of any one of claims 58 to 65, wherein the first temperature condition is in the range of about 80°C to about 100°C.

67. The computer readable medium of any one of claims 58 to 66, wherein the second temperature condition and / or time period is associated with an annealing phase or an extension phase of the PCR process.

68. The computer readable medium of any one of claims 58 to 67, wherein the first temperature condition and / or time period is associated with a denaturation phase of the PCR process.

69. The computer readable medium of any one of claims 58 to 68, wherein the PCR process comprises a plurality of PCR cycles.

70. The computer readable medium of claim 69, wherein the first probe type is associated with a cumulative emission signal that remains substantially stable during multiple stages of PCR cycling and that increases cumulatively over multiple PCR cycles.

71. The computer readable medium of any one of claims 69 to 70, wherein the second probe type is associated with a transient emission signal that fluctuates over multiple stages in each PCR cycle.

72. The computer-readable medium of any one of claims 58 and 59, wherein the first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, respectively.

73. A computer-readable medium according to any one of claims 58 and 59, wherein the obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an endpoint cycle of the PCR process, wherein the first set of reaction conditions and the second set of reaction conditions are different from each other.

74. The computer readable medium of any one of claims 58 and 59, wherein the amplification process is an endpoint PCR process.

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