Systems and methods for analyte detection in multiplexed assays

Abstract

Systems and methods that enable analyte detection in a multiplexed amplification process may include obtaining, at multiple time points during the amplification process, composite luminescence signal data associated with a composite luminescence signal from at least a first probe type including a first label configured to generate a first luminescence signal and a second probe type including a second label configured to generate a second luminescence signal having spectrally similar characteristics to the first luminescence signal, wherein the first probe type and the second probe type have different thermal and / or temporal characteristics; and determining, based at least in part on the composite luminescence signal data, luminescence signal data associated with a luminescence signal from a given probe type of the first probe type or the second probe type during the amplification process.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 356,874, entitled "Systems and Methods for Enabling Multiplexed Polymerase Chain Reaction Processes," filed June 29, 2022; U.S. Provisional Patent Application No. 63 / 356,863, entitled "Compositions and Methods for Detecting Nucleic Acids Using Intra-Channel Multiplexing," filed June 29, 2022; and U.S. Provisional Patent Application No. 63 / 408,665, entitled "Compositions and Methods for Detecting Nucleic Acids Using Intra-Channel Multiplexing," filed September 21, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION Aspects of the present disclosure relate to systems and methods for analyte detection in multiplexed assays. In particular, the present disclosure relates to nucleic acid detection using multiplexed nucleic acid amplification assays, such as polymerase chain reaction assays. [Background technology]

[0003] Introduction Nucleic acid detection assays are often performed by adding a sample suspected of containing one or more target nucleic acids to a reaction mixture. The reaction mixture may contain one or more detectable labels, each designed to associate with a different target nucleic acid and generate a signal corresponding to the amount of the relevant target nucleic acid in the reaction mixture. In a "singleplex" assay, the reaction mixture contains a single detectable label designed to associate with a single target nucleic acid. Conversely, in a "multiplex" assay, the reaction mixture typically contains multiple different detectable labels, each designed to be specific for a different target nucleic acid.

[0004] Thus, multiplex assays allow for the detection of multiple different targets in a single reaction mixture. In some applications, the detectable label is a fluorescent dye that is incorporated into a nucleic acid probe, primer, or some other nucleic acid molecule designed to specifically hybridize with the corresponding target nucleic acid that it is designed to associate with.

[0005] When implementing multiplex systems and processes for determining the relative amounts of different target nucleic acids in a sample, several challenges can arise. In particular, the use of detectable labels that have spectral similarity (emit identical or overlapping luminescence signals) can make it difficult to determine the contribution of each label individually and, therefore, each different target nucleic acid with which they are associated.

[0006] For example, there is a need to provide more robust systems and methods for performing multiplexed nucleic acid detection assays, such as nucleic acid detection utilizing various polymerase chain reaction (PCR) assays, and for analyzing data associated with such assays. [Brief explanation of the drawings]

[0007] Various objects, features, characteristics and advantages of the inventions within the scope of the present disclosure will become apparent and will be more readily understood when taken in conjunction with the following description of various embodiments in conjunction with the accompanying drawings and claims, all of which form a part of this specification. In the drawings, like reference numerals may be utilized to designate corresponding or similar parts in the various views, and the various elements shown are not necessarily drawn to scale. [Figure 1] 1 shows the emission spectra of various fluorescent dyes that can be used in nucleic acid detection assays. [Figure 2A] FIG. 1 is a schematic diagram of an approach for enabling detection of multiple target nucleic acids using spectrally similar detectable labels, according to various embodiments of the present disclosure. [Figure 2B] 2B is a graph showing signal response over time for the approach outlined in FIG. 2A, according to various embodiments of the present disclosure. [Figure 3A] 1 shows the activity of cleavable and non-cleavable probes during the annealing, extension, and denaturation steps of thermal cycling according to an embodiment of the present disclosure. [Figure 3B] 3B is a graph showing fluorescent signal response over time during thermal cycling of an amplification process utilizing the cleavable and non-cleavable probes of FIG. 3A according to an embodiment of the present disclosure. [Figure 4] FIG. 1 shows a flow diagram illustrating exemplary operations associated with enabling analyte detection in a multiplexed amplification process according to an embodiment of the present disclosure. [Figure 5A] Shown are the results of dual assay studies in which TaqMan probes and extendible fluorogenic (EF) probes were designed to generate spectrally similar fluorescent signals (Figure 5A) or to generate fluorescent signals with different spectral profiles (e.g., no significant overlap) (Figure 5B). [Figure 5B]Shown are the results of dual assay studies in which TaqMan probes and extendible fluorogenic (EF) probes were designed to generate spectrally similar fluorescent signals (Figure 5A) or to generate fluorescent signals with different spectral profiles (e.g., no significant overlap) (Figure 5B). [Figure 5C] The EF-associated fluorescent signal derived using the results of the assay in Figure 5A is compared with the EF-associated fluorescent signal measured directly in the assay in Figure 5B. [Figure 5D] The fluorescent signals of the TaqMan probe and the extendible fluorogenic probe (EF) during the extension and denaturation steps are shown. [Figure 6] 1 shows the results of another 9-plex assay test containing five different detectable labels, in which the corresponding TaqMan probe and EF probe share four of the detectable labels, and one of the detectable labels is associated only with the TaqMan probe, according to embodiments of the present disclosure. [Figure 7A] 1 illustrates the process of using a tailed primer specific for a nucleic acid target to form a template to which an EF probe can hybridize. [Figure 7B] 7A-7C show exemplary tailed forward primers, reverse primers, and EF probes that may be included in a reaction mixture for carrying out the process of FIG. 7A. DETAILED DESCRIPTION OF THE INVENTION

[0008] In the context of nucleic acid probes and target nucleic acids, the term "specifically interacting" (and similar terms) refers to a probe designed to interact with a target to a greater extent than with non-target nucleic acids also present in the reaction mixture. For example, specific interaction can include full or partial hybridization of the probe with its corresponding target. Hybridization between the probe and the target need not be 100%. For example, functionally effective interaction can be achieved using probes having 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 their respective targets.

[0009] As used herein, a "detection channel" refers to a specific subset of the full range of possible values of a detectable signal. For example, if the detectable signal is a fluorescent signal, a detection channel (e.g., a fluorescent channel or dye channel) may represent a wavelength band of a specific size. A detection channel may have a band size of approximately 10-60 nm, depending on, for example, the sensitivity of the instrument and / or the desired signal resolution accuracy. A detection channel may also include discrete wavelengths or wavelength ranges. Additionally or alternatively, a detection channel may be defined according to the optical filter arrangement used to measure the detectable signal. Each different detection channel typically includes a specific optical filter arrangement to block emissions outside of that channel. Thus, as a functional definition, each detectable signal within a given optical filter arrangement may be considered to be within the same detection channel.

[0010] As used herein, distinct fluorescent signals having "substantially identical fluorescence" provide fluorescent emissions within similar wavelength bands. For example, a first fluorescent signal and a second fluorescent signal having substantially identical fluorescence may have emission peaks that differ by about 10 nm or less, or about 8 nm or less, or about 6 nm or less, or about 4 nm or less, or about 2 nm or less, or about 1 nm or less, or may have emission peaks that are substantially indistinguishable from one another based on the sensitivity of the detection instrument used to measure the fluorescent emissions. Additionally or alternatively, fluorescent signals may be considered to have "substantially identical fluorescence" in applications where they are measured using the same optical filter arrangement.

[0011] As used herein, a detectable signal having a "substantial signal" and / or "substantial fluorescence" refers to a signal significantly above background (i.e., baseline) levels, including fluorescent signals significantly above background / baseline levels of fluorescence. This may be determined by a threshold value that separates substantial fluorescence from background fluorescence. The threshold value may vary according to the needs of a particular test protocol and application. In some embodiments (e.g., embodiments without a passive reference), the threshold value is set, for example, between about 1,000 and about 30,000, or more typically between about 2,000 and about 20,000, or between about 3,000 and about 15,000, or between about 4,000 and about 6,000, or within a range having endpoints defined by any two of the above values. In some embodiments (e.g., embodiments with a passive reference), the threshold value is set, for example, between about 0.01 and 0.5 of the change in fluorescent signal from a detrended baseline (ΔRn). In some embodiments, the threshold is some percentage higher than the baseline level, such as about 5 percent to about 10 percent higher than the baseline level.

[0012] The "background" or "baseline" level of signal during the amplification process (i.e., 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 value of the amplification cycle before exponential amplification occurs. For example, exponential amplification can be determined when the change in signal from one amplification cycle to the next exceeds a certain percentage, indicating exponential change.

[0013] As a result, signal and / or fluorescence levels that are not "substantial" in accordance with the above may be described herein as "negligible." Similarly, with respect to probe binding, a probe "substantially binds" to its target when it binds significantly above background (e.g., above binding to non-targets). Optionally, at least 1%, 5%, 10%, 20%, 50%, or 80% of the probe or target binds.

[0014] As used herein, a "cleavable" probe is one that is intended to be cleaved as a result of specific interaction of the probe with its respective target and the corresponding label is released, resulting in a corresponding increase in detectable signal.

[0015] As used herein, a "non-cleavable" probe refers to a probe having a label that is intended to remain associated with the probe throughout the assay. In a non-cleavable probe, the corresponding detectable signal does not change due to release of the label from the probe, but rather changes according to changes in the structure of the probe. An extendable fluorogenic probe is an example of a non-cleavable probe. For example, an extendable fluorogenic probe may include a universal or hairpin extendable fluorogenic probe, as described in various embodiments, or may have a non-hairpin structure.

[0016] The terms "detectable signal" and "label signal" are used interchangeably herein. For example, a "first label signal" refers to the signal emitted by a first label of a first probe type, and a "second label signal" refers to the signal emitted by a second label of a second probe type. A "total signal" refers to the entire signal measured in a particular detection channel at a given time or measurement point. Multiple 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 instances, a "total signal" may be considered 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" may be used as specific examples of the corresponding broader terms.

[0017] The term "spectral similarity" refers to the emission signals of detectable labels having the same or substantially overlapping spectral profiles. Thus, different probe types carrying the same detectable label or different detectable labels whose emission signals exhibit substantial spectral overlap can both be considered spectrally similar probes. In embodiments, spectrally similar detectable labels may be detectable in the same optical detection channel, although other techniques may be used to detect the emission signals of such detectable labels as well. Reference to substantially overlapping spectra should be understood to mean spectral similarity. While spectral similarity refers to emission signals exhibiting the same or somewhat overlapping spectral signals and profiles, the term does not refer to the time course or period during which such signals with corresponding profiles are emitted, such as during different stages of an amplification process.

[0018] "Endpoint," when referring to cycles, refers to the designated cycle at which the PCR process is considered complete and / or the designated cycle at which a signal threshold above background signal occurs by a predetermined amount. In various embodiments, the endpoint cycle according to the present disclosure can be in the range of 20 to 45 cycles, e.g., 30 to 40 cycles. However, the number of cycles until the endpoint cycle can differ from these ranges and can be the designated cycle and / or the cycle correlating to when a luminescence (e.g., fluorescence) signal indicative of an amplification product reaches a predetermined level. Additionally, "endpoint signal" refers to the luminescence signal measured at the endpoint cycle.

[0019] The terms "determining," "calculating," and "estimating" are used interchangeably herein. These terms are not intended to imply an exact level of measurement precision. Thus, when values are "determined," "calculated," or "estimated" using the embodiments described herein, it will be understood that such values may contain some inherent error due to factors such as tolerances of detection equipment, rounding, variations in chemical reactions, and other inherent measurement imperfections that are known and understood by those skilled in the art.

[0020] Furthermore, for any given element of a component of a described embodiment, unless stated otherwise, either implicitly or explicitly, any of the possible alternatives listed for that element or component may generally be used individually or in combination with each other.

[0021] Additionally, unless otherwise indicated, numbers expressing quantities, components, distances, or other measurements used in the specification and claims are to be understood as being optionally modified by the term "about" or its equivalents. When terms such as "about," "approximately," or "substantially" are used in connection with a stated quantity, value, or condition, they may be interpreted to mean an amount, value, or condition that deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% from the stated quantity, value, or condition. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0022] The headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0023] It should also be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" do not exclude plural referents unless the context clearly dictates otherwise. Thus, for example, embodiments that refer to a singular referent (e.g., a "widget") may also include two or more such referents.

[0024] It will also be understood that the embodiments described herein may include properties and / or characteristics (e.g., components, components, members, elements, parts, and / or portions) described in one or more separate embodiments, and are not necessarily limited to the precise features explicitly described for that particular embodiment. Accordingly, various features of a given embodiment can be combined with and / or incorporated into other embodiments of the present disclosure. Thus, the disclosure of a particular feature in connection with a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of that feature to the particular embodiment. Rather, it will be understood that other embodiments may also include such features.

[0025] Overview of multiplexing using probes with spectrally similar detectable labels In various multiplex nucleic acid detection assays, detectable labels are assigned to different targets. The presence and / or amount of each target can then be determined, for example, by measuring the signals emitted from the detectable labels in separate "detection channels," each corresponding to a specific characteristic of the corresponding emitted signal. For example, in the context of fluorescent dyes as detectable labels, separate detection channels may correspond to the emission wavelength spectra associated with each dye. However, there may be a certain amount of overlap in the emission spectra of different dyes. As the overlap (spectral similarity) in the emission spectra increases, it becomes more difficult to resolve the separate detected fluorescent emission signals, and therefore, it becomes more difficult to detect and / or quantify each target.

[0026] Although multiplexing dyes can be selected with the aim of minimizing spectral overlap, the finite emission spectra practically limit the number of distinct dyes that can be combined in the same multiplex assay. As a result, there is currently a significant constraint on the number of different targets that can be detected and / or measured in a multiplex assay. Therefore, there remains a need for compositions and methods that can increase the "plexity" of detection assays. Furthermore, it may be desirable to use detectable labels with spectral similarity, for example, using dyes with some overlap in emission spectra, and / or to use the same dye for different target nucleic acids.

[0027] Various embodiments disclosed herein relate to systems and methods for enabling multiplexed nucleic acid detection assays that rely on the polymerase chain reaction (PCR) process by enabling the determination of distinct detectable signals, each associated with a different assay target nucleic acid but having spectral similarity. For example, various embodiments enable the detection of spectrally similar detectable labels within the same detection channel (e.g., within a channel sensitive to emissions (e.g., fluorescence emissions) within a defined spectral range).

[0028] FIG. 1 shows the emission spectra 150 of various fluorescent dyes 152 that can be used in nucleic acid detection assays. As described above, a multiplex assay can determine the presence and / or amount of each target by assigning each dye as a label for a distinct target nucleic acid and then measuring the fluorescent signal in a separate detection channel, each corresponding to a different emission wavelength of the corresponding dye. As shown, there can be some overlap (e.g., a substantial amount) in the emission spectra 150 of one or more of the dyes 152 such that the dyes are spectrally similar. For example, as shown in FIG. 1, AF647 and Cy5 dyes have substantially the same emission spectrum and therefore have spectral similarity. While multiplexing dyes are typically selected with the goal of minimizing spectral overlap, the finite nature of emission spectra practically limits the number of distinct dyes that can be combined in the same multiplex assay, and therefore serves as a practical limit to the number of distinct targets that can be detected and / or measured without increasing the complexity of the system, such as by increasing the number of distinct detection channels and / or implementing other relatively complex deconvolution schemes.

[0029] Various embodiments described herein solve one or more of the aforementioned problems by enabling analyte detection in a multiplexed amplification process by utilizing multiple detectable signals, each detectable signal associated with a different assay target analyte or set of target analytes, having spectral similarity in their emission spectra, and / or capable of being detected using the same detection channel. The multiple detectable signals can be resolved separately and analyzed independently, thereby enabling detection and / or quantification of each target. By enabling assay of multiple target analytes utilizing the same detectable label (e.g., dye) or detectable labels with spectral similarity, the disclosed embodiments can beneficially increase "multiplexity" (i.e., the number of target analytes that can be detected and quantified in a multiplexed assay) without relying on problems associated with additional detectable labels (e.g., dyes), detection channels, and / or spectral overlap. For example, in accordance with aspects of the present disclosure, a common detection channel can be used to detect labels with spectral similarity but targeted to different target analytes. Similarly, embodiments described herein can beneficially reduce the number of distinct detectable labels (e.g., dyes) required for a multiplex assay without reducing the assay's multiplexity. Furthermore, various embodiments can enable the use of the same detectable label (e.g., dye) as a label for different target nucleic acids in a multiplex assay, including using the same target for different target nucleic acids in a multiplex assay, including within the same cycle of a PCR reaction. Furthermore, various embodiments can enable detection of the same dye in the same detection channel.

[0030] 2A is a schematic diagram of a technique for detecting multiple target nucleic acids using spectrally similar detectable labels by preparing different first and second probe types, varying reaction mixture conditions, and measuring the total signal obtained 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 capable of generating a first label signal 214. A 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 capable of generating a second label signal 216.

[0031] In some embodiments, the first label 210 and the second label 212 are the same. For example, the first label 210 and the second label 212 may comprise the same fluorescent dye. In some embodiments, the first label 210 and the second label 212 may be different but are still designed to generate emission signals having substantially the same or some overlapping spectral profile (e.g., having spectral similarity). For example, the first label 210 and the second label 212 may be chemically distinct but comprise dyes that function to emit signals having similar wavelengths. In some embodiments, the first label signal 214 and the second label signal 216 are measured using the same detection channel (e.g., one that includes an optical filter arrangement) within a detection instrument.

[0032] A first probe 202 and a second probe 204 may be prepared in the same reaction mixture and allowed to specifically interact with any first targets 206 and second targets 208, respectively, in the reaction mixture. As shown, the reaction mixture is subjected to at least two different reaction conditions. The first probe 202 is designed so that the first label 210 generates a first label signal 214 to an extent proportional to the amount of specific interaction between the first probe 202 and the first target 206 under both a first set of conditions 218 and a second set of conditions 220. In contrast, the second probe 204 is designed so that the second label 212 generates a second label signal 216 to an extent proportional to the amount of specific interaction between the second probe 204 and the second target 208 under the second set of conditions 220 but not under the first set of conditions 218. In other words, under the first set of conditions 218, the first label signal 214 increases as a result of the specific interaction between the first probe 202 and the first target 206, but the second label signal 216 is not emitted as a result of the specific interaction between the second probe 204 and the second target 208. Under the second set of conditions 220, the second label signal 216 increases as a result of the specific interaction between the second probe 204 and the second target 208, and the first label signal 214 also increases further or remains at a relatively high level, at least to some extent from the first set of conditions.

[0033] In the first set of conditions 218, the second label 212 does not produce a "substantial signal" (e.g., fluorescence), and therefore the second label signal 216 is not substantially different from the background (i.e., baseline) level of luminescent signal (e.g., fluorescence) in the reaction mixture. That is, in the first set of conditions 218, although there may be some non-zero level of signal produced by the second label 212, the second label signal 216 typically remains below the threshold that separates a meaningful signal from a background signal. This threshold can vary according to the needs of a particular test protocol and application, as described above.

[0034] In at least some embodiments, when both the first target 206 and the second target 208 are present in the reaction mixture, the second label signal 216 differs more between the first set of conditions 218 and the second set of conditions 220 than the first label signal 214 differs between the first set of conditions 218 and the second set of conditions 220. Thus, while the first label signal 214 may differ to some extent between the first set of conditions 218 and the second set of conditions 220, this difference is typically less than the difference in the second label signal 216 between the first set of conditions 218 and the second set of conditions 220.

[0035] Various embodiments of the present disclosure utilize the difference in how the first label signal 214 and the second label signal 216 respond to different sets of conditions to allow the detected first label 214 and second label signal 216 to be resolved (separated), even when they are detected within the same detection channel (e.g., a given optical filter arrangement that filters a defined emission spectrum within the channel). As shown, a total signal (or composite signal) ("first total signal") is measured under a first set of conditions 218, and a signal ("second total signal") is measured under a second set of conditions 220. Fluorescent signal data representing the first total signal may be referred to herein as "first fluorescent signal data," and fluorescent signal data representing the second total signal may be referred to herein as "second fluorescent signal data" or "composite fluorescent signal data." As used herein, first and second in this context are not necessarily used to indicate a temporal order of detections or conditions, although such a temporal order may occur.

[0036] In the first set of conditions 218, the total signal is substantially equal to the first label signal 214. That is, the first total signal is primarily composed of the first label signal 214, with a negligible contribution from the second label signal 216. In the second set of conditions 220, the total signal includes a combination of the first label signal 214 and the second label signal 216. Thus, the first label signal 214 and the second label signal 216 can be resolved separately based on the first total signal and the second total signal. For example, the first label signal 214 can be determined based on the first total signal, and the second label signal 216 can be resolved by subtracting the first total signal from the second total signal.

[0037] In some embodiments, the first label signal 214 is directly equal to the first total signal. In other embodiments, the first label signal 214 is determined as a function of the first total signal. In some embodiments, this function is linear (although in some embodiments, a nonlinear function may be used). For example, as noted above, the first label signal 214 may differ slightly between the first set of conditions 218 and the second set of conditions 220, even when the amount of the first target 206 remains unchanged. In certain applications, the first label signal 214 under the second set of conditions 220 may better correspond to a standard curve in which the first label signal 214 is equal to the amount of the first target 206. Thus, by estimating the first label signal 214 as a function of the first total signal rather than directly equal to the first total signal, the calculated first label signal 214 can be closer to that measured under the second set of conditions 220 (i.e., in the absence of the interfering second label signal 216).

[0038] In some embodiments, the function for converting the first total signal to 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 signals 214 under the first set of conditions 218 and the second set of conditions 220 may be correlated to each other according to a linear function. In other embodiments, a non-linear function may be used to correlate them. Using a linear function, a multiplier (e.g., a correction factor) may be used to convert the first total signal to the first label signal 214. Once such a linear function is determined, it can be used in subsequent assays without necessarily needing to further compare the first label signals 214 under the first set of conditions 218 and the second set of conditions 220 in the absence of the second probe having the second target. As noted above, in some embodiments, the function for converting the first total signal to the first label signal may be non-linear. In some embodiments, the function / correlation is determined across or between thermal cycle stages where the number of cleaved probes is expected to be the same. This approach can be used to resolve different signals, even when detected, for example, in the same detection channel.

[0039] As explained in more detail below, the first probe 202 and the second probe 204 have different mechanisms of action that allow for different signal responses to the first set of conditions 218 and the second set of conditions 220 depending on the probe type. Beneficially, attributes other than different melting temperatures of the probes may be relied upon to allow for the resolution of the distinct signals associated with each of the different probe types. Thus, while the first probe 202 and the second probe 204 may have different melting temperatures, such different melting temperatures are not a requirement for their associated label signals to be effectively resolved. In some embodiments, for example, the melting temperature (T m ) and T of the second probe 204 mare within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other, although such melting temperature differences are not intended to limit the scope of the present disclosure. Furthermore, given the techniques that allow for the differentiation of signal responses according to embodiments of the present disclosure, there is no need to rely on the melting step of the amplification process.

[0040] 2B is a graph that schematically illustrates signal response over time based on cycling a reaction mixture between a first set of reaction conditions 218 and a second set of reaction conditions 220, and the presence of both a first target 206 and a second target 208 in the reaction mixture, for the approach outlined in FIG. 2A. The cycling of conditions can include, for example, different conditions for various stages associated with thermal cycling in a nucleic acid amplification reaction, such as PCR. In such a reaction, the first set of reaction conditions 218 corresponds to supporting the denaturation stage of thermal cycling, and the second set of reaction conditions 220 corresponds to supporting the annealing and / or extension stage ("annealing / extension stage") of 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).

[0041] As shown, under the second set of reaction conditions 220, both the first label signal 214 and the second label signal 216 increase. Under the first set of reaction conditions 218, the first label signal 214 remains approximately the same as at the end of the previous cycle (although it may vary slightly as described above), while the second label signal 216 decreases to a level equivalent to the baseline signal level of the second label signal 216, which may be substantially constant over multiple amplification cycles. In other words, the second label signal 216 exhibits a baseline signal above the background signal level under the first set of reaction conditions. In some cases, the second label signal may exhibit a baseline signal level that varies at different stages of the amplification cycle, but is nevertheless sufficiently distinguishable from and lower than the level under the second set of reaction conditions. This may be due to the different state of the probe and the proximity of the quencher and label.

[0042] As shown, both the first label signal 214 and the second label signal 216 cumulatively increase with each subsequent occurrence of the second set of conditions 220. This is the result of additional specific interactions occurring between the first probe 202 and the first target 206 in the reaction mixture, and additional specific interactions occurring between the second probe 204 and the second target 208 in the reaction mixture. However, while the first label signal 214 remains at a similar level when transitioning from the end of one cycle to the beginning of another (i.e., when transitioning 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 next cycle), the second label signal 216 returns to near-baseline levels at the beginning of each cycle (i.e., when the first set of conditions 218 occurs). In other words, the first label signal 214 is continuous and cumulative over subsequent cycles, while the second signal is transient and dependent on the set of conditions occurring during the cycle.

[0043] Although some embodiments described herein may be utilized with intra-channel multiplexing (detection of labels in the same detection channel), the present disclosure is not limited thereto. Furthermore, the present disclosure also contemplates intra-channel multiplexing in combination with inter-channel multiplexing to further increase assay multiplexity. For example, an assay may be designed with multiple different detectable labels (e.g., dyes) having spectra for detection using multiple different detection channels, one or more of which are configured to detect multiple detectable signals of signal responses from different targets using spectrally similar detectable signals that can be resolved according to the techniques described herein. Furthermore, by utilizing aspects of probes with detectable labels designed to interact and exhibit different temporal emission signal patterns, it may be possible to use additional probe types in conjunction with the above probe types and perform measurements at additional time periods throughout the reaction cycle, allowing further identification of which signals correspond to which target nucleic acids.

[0044] Cleavable and non-cleavable probes In some embodiments, the first probe (e.g., first probe 202) is a "cleavable" probe. The first probe can be designed such that, upon hybridization of the first probe with a first target (e.g., first target 206), a first label (e.g., first label 210) is separated from the first probe (and released, e.g., from a corresponding quencher). Thus, after being released, the first label continues to contribute to the total signal in the reaction mixture, thereby generating a cumulative luminescent signal. The first probe can be, for example, a TaqMan probe that undergoes cleavage as a result of the 5' to 3' exonuclease activity of a DNA polymerase during extension of the target molecule to which the probe is hybridized. 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, and 5,8 Nos. 01,155, 5,804,375, 5,876,930, 5,994,056, 6,030,787, 6,084,102, 6,127,155, 6,171,785, 6,214,979, 6,258,569, 6,814,934, 6,821,727, 7,141,377, and 7,445,900, all of which are incorporated herein by reference.

[0045] 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 signal generated varies depending on the structure of the probe rather than on the release of the label. The second probe may be, for example, an extendible fluorogenic (EF) probe, which quenches the label when in a single-stranded structure but allows for signaling (i.e., generates a transient luminescent signal) when incorporated into a double-stranded molecule.

[0046] The EF probe can be, for example, a universal extendable fluorogenic probe, an extendable hairpin probe designed for specific target amplification, or an extendable probe having a non-hairpin sequence structure.

[0047] 3A illustrates the activity of cleavable probe 302 (which in various embodiments may be a TaqMan probe) and non-cleavable probe 312 (which in various embodiments may be an EF probe) during the annealing, extension, and denaturation phases of a PCR reaction. As shown, during the annealing phase, TaqMan probe 302 hybridizes to its corresponding target nucleic acid amplicon 304 (as used herein, target nucleic acid amplicon may refer to one strand of a target double-stranded nucleic acid and should be understood by reference to the context in describing a PCR reaction). During extension of primer 303 hybridized to target nucleic acid amplicon 304 upstream of probe 302, the 5' to 3' exonuclease activity of the DNA polymerase cleaves TaqMan probe label 306 from the remainder of probe 302, thereby separating it from the corresponding TaqMan probe quencher 309. This results in a corresponding increase in fluorescent signal. During the denaturation process, the label 306 remains free in the reaction mixture solution and therefore continues to contribute to the total fluorescent signal.

[0048] EF 312 comprises an EF label 316 and an EF quencher 319, which remain in close proximity to each other while the extendable probe 312 is in a single-stranded conformation. Thus, the fluorescent signal from the label 316 remains substantially quenched while the EF is in a single-stranded conformation. During the annealing and extension steps, EF 312 hybridizes to its corresponding target template amplicon 314 and is extended to form an extender probe amplicon 313. Extension of the target template 314 then forms a complement 315 of the extender probe amplicon 313. The resulting double-stranded amplicon 317 separates the label 316 from the quencher 319 far enough to allow fluorescence emission. During denaturation, the extender probe amplicon 313 separates from its complement 315. Upon return to a single-stranded conformation, the label 316 and quencher 319 return to close proximity, and fluorescence is again quenched.

[0049] FIG. 3B is a graph showing the fluorescent signal over time from TaqMan probe 302 and EF probe 312 during thermal cycling of the amplification process. The temperature of the thermal cycling may vary depending on the needs of a particular application. By way of example, the denaturation step may be performed at a temperature in the range of about 80°C to about 100°C, e.g., about 85°C to about 95°C, e.g., about 90°C to about 95°C. The annealing / extension step may be performed at a lower temperature, e.g., about 40°C to about 75°C, e.g., about 50°C to about 70°C, e.g., about 55°C to about 65°C. In an embodiment, a first set of reaction conditions (e.g., first set of conditions 218 described with reference to FIG. 2A) corresponds to denaturation step 318, and a second set of reaction conditions (e.g., second set of conditions 220 described with reference to FIG. 2A) corresponds to annealing / extension step 320.

[0050] While various embodiments cycle between a denaturation step 318 and a combined annealing / extension step 320 (i.e., the amplification process cycles between two target temperatures), other embodiments may include separate annealing and extension steps. In such embodiments, the temperature and possibly other reaction conditions may be varied between the annealing and extension steps. For example, the extension step may be performed at a temperature higher than that of the annealing step. In some embodiments, the amplification process cycles between two different target temperatures or two different target temperature ranges 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 amplification process cycles.

[0051] 3B shows that the fluorescent signal associated with TaqMan probe 302 increases during extension phase 320 and then remains at a similar level throughout the denaturation phase 318 of the next cycle (although some relatively insignificant decrease in signal may occur as described above), whereas the fluorescent signal associated with EF probe 304 increases during extension phase 320 but decreases to the baseline signal level associated with EF probe 304 once the subsequent denaturation phase 318 reaches the target denaturation temperature. Those skilled in the art will understand that cycle N, cycle N+1, and cycle N+2 in FIG. 3B may start from different phases, in which case the comparison of the signal levels described above may be shifted.

[0052] In some embodiments, the first set of reaction conditions (e.g., denaturing conditions 318) includes a first measurement temperature at which a first label signal is measured, and the second set of reaction conditions (e.g., annealing / extension conditions 320) includes a second, different measurement temperature at which a first label signal and a second label signal are measured. In some embodiments, the first and second measurement temperatures differ by at least about 10°C, about 15°C, about 20°C, about 25°C, or about 30°C. The first measurement temperature can be a target denaturing temperature in the range of, for example, about 80°C to about 100°C, e.g., about 85°C to about 95°C, e.g., about 90°C to about 95°C, and the second measurement temperature can be a target annealing / extension temperature in the range of, for example, about 40°C to about 75°C, e.g., about 50°C to about 70°C, e.g., about 55°C to about 65°C.

[0053] Exemplary Techniques and Implementations The disclosed techniques for enabling analyte detection in a multiplexed amplification process may involve the performance of a variety of operations. For example, Figure 4 shows an exemplary flow diagram 400 illustrating operations associated with enabling analyte detection in a multiplexed amplification process (e.g., a multiplexed polymerase chain reaction (PCR) process).

[0054] Operation 402 of flow diagram 400 includes obtaining, at one or more time points during one or more cycles of an amplification process, luminescence (e.g., fluorescence) signal data related to a combined luminescence (e.g., fluorescence) signal from at least a first probe type including a first detectable label (e.g., a fluorophore) and a second probe type including a second detectable label (e.g., a fluorophore) having spectral characteristics 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 first probe 202 and second probe 204 described above with reference to FIG. 2A). For example, the first probe type and the second probe type may have different thermal and / or temporal characteristics. As described above, the first detectable label and the second detectable label may have spectral similarity such that both may be configured to generate luminescence signals (e.g., fluorescence) having substantially similar ranges of wavelengths (e.g., corresponding to first label 212 and second label 212 described above with reference to FIG. 2A).

[0055] The composite signal may include luminescence (e.g., fluorescent) signals substantially contributed by both the first probe type and the second probe type. As noted above, luminescence (e.g., fluorescent) signal data associated with such a composite signal (and captured during particular reaction conditions, such as particular temperature conditions) may be considered "composite luminescence signal data" or "second luminescence signal data" or "composite fluorescent signal data" or "second fluorescent signal data."

[0056] As discussed in more detail below, Figure 5A provides an exemplary graphical depiction of composite fluorescent signal data from a first probe type and a second probe type (e.g., TaqMan and EF probes are labeled with a FAM fluorophore, and the FAM signal at 64°C (top row) represents the composite fluorescent signal). In some instances, 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, both the TaqMan and EF probes are FAM-labeled). Despite potential commonality between the fluorophores of the two probe types, the two probe types can be configured differently to have respective binding affinities for different analytes (e.g., different target nucleic acids). For example, a first probe type may have a binding affinity for a first analyte (e.g., corresponding to a first probe 202 designed to interact with a first target 206, as described above with reference to Figure 2A), and a second probe type may have a binding affinity for a second analyte that is different from the first analyte (e.g., corresponding to a second probe 204 designed to interact with a second target 208, as described above with reference to Figure 2A).

[0057] In some implementations, one probe type (e.g., the first probe type) is a cleavable probe and the other probe type (e.g., the second probe type) is non-cleavable. In some implementations, the cleavable probe type is a TaqMan probe and the non-cleavable probe type is an EF probe (e.g., in the example described with reference to Figures 3A, 3B, and 5A-5D). The first probe type may be associated with a cumulative fluorescence that remains substantially stable over multiple segments of the amplification cycle and that increases cumulatively over multiple amplification cycles, while the second probe type may be associated with a transient fluorescence that fluctuates significantly over each amplification cycle, such as during different stages of the amplification cycle.

[0058] Operation 404 of flow diagram 400 includes determining luminescence (e.g., fluorescence) signal data associated with the luminescence (e.g., fluorescence) signal from a given probe type during one or more cycles of the amplification process based at least in part on the luminescence (e.g., fluorescence) signal data associated with the composite luminescence signal and the thermal and / or temporal characteristics of the first probe type and / or the second probe type. The given probe type to which the determined luminescence (e.g., fluorescence) signal is associated may include at least one of the first probe type or the second probe type (e.g., the derived EF-related signal of FIG. 5C).

[0059] In some implementations, the first luminescent signal data is acquired at multiple time points during the amplification process resulting in composite luminescent signal data. The first luminescent signal data may be associated with different reaction conditions than the reaction conditions associated with the composite luminescent signal data. For example, the composite luminescent signal data may be associated with a second set of reaction conditions (e.g., corresponding to the second set of conditions 220, 320 described above with reference to Figures 2A, 2B, and 3B), and the first luminescent signal data may be associated with a first set of reaction conditions (e.g., corresponding to the first set of conditions 218, 318 described above with reference to Figures 2A, 2B, and 3B).

[0060] In some examples, the 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 is associated with first luminescence signal data and a second temperature condition is associated with second or composite luminescence signal data). In some examples, the first temperature condition is higher than and / or does not overlap with the second temperature condition. For example, FIG. 3B shows a second temperature condition of 65°C that may be associated with composite luminescence signal data and a first temperature condition of 95°C that may be associated with second luminescence signal data. Other temperature conditions are within the scope of the present disclosure (e.g., a second temperature condition in the range of about 40°C to 75°C, a first temperature condition in the range of about 80°C to 100°C). The first and second temperature conditions may be associated with different stages of the amplification process. For example, the second temperature conditions may be associated with the annealing or extension stage, or a combined extension / annealing stage, of the amplification process (e.g., the second set of conditions 320 of the various amplification cycles depicted in Figure 3B), while the first temperature conditions may be associated with the denaturation stage of the amplification process (e.g., the first set of conditions 318 of the various amplification cycles depicted in Figure 3B).

[0061] Therefore, taking into account reaction differences in reaction conditions, the composite and first luminescent signal data may be associated with different time points or periods within the amplification process. For example, the data points (or subsets of data points) forming the composite luminescent signal and the first luminescent signal data may be acquired / captured in a time-interleaved manner (e.g., data points for the composite luminescent signal data and the first luminescent signal data alternate as reaction conditions are alternated during the amplification process). Furthermore, the first luminescent signal and the composite luminescent signal may be captured by collecting several data points over the course of an amplification (e.g., PCR) cycle and separating the two signals according to their different temporal characteristics / profiles. As noted above, this approach may also be utilized with three or more probe types with detectable labels by utilizing the different temporal aspects of the various luminescent signals.

[0062] Determining the luminescence signal data associated with the luminescence signal of a given probe type may utilize as input the composite luminescence signal data (described above with reference to operation 402) and the first luminescence signal data, as described in more detail below with reference to FIG. 5A (e.g., with brief reference to FIG. 5A , the signal data in the top left column represent exemplary measured composite fluorescent signal data, the signal data in the middle left column represent exemplary converted first fluorescent signal data determined using the measured first fluorescent signal data, and the signal data in the bottom left column represent exemplary fluorescent signal data associated with the fluorescent signal of a given probe). The fluorescent signal data associated with the fluorescent signal of a given probe can be determined in real time during the amplification process or as a post-processing operation.

[0063] Figures 5A and 5B show the results of qPCR duplex assays in which the TaqMan and EF probes were designed to have spectral similarity, e.g., to generate detectable fluorescent signals in the same dye channel (Figure 5A), or to not exhibit significant spectral overlap, e.g., to generate distinct fluorescent signals detectable in different dye channels (Figure 5B). In the assay shown in Figure 5A, both the TaqMan and EF probes were labeled with FAM; therefore, the VIC fluorophore signal (shown in the right-hand column of the data results in Figure 5A) was used as a control. In the assay shown in Figure 5B, the TaqMan probe was labeled with VIC, and the EF was labeled with FAM. The reaction mixture composition, template DNA concentration, and amplification conditions were otherwise kept the same between the two assays.

[0064] In Figure 5A, graph 502 shows the change in FAM fluorescence signal (ΔRn) (y-axis) from which the baseline signal has been detrended versus the cycle number (x-axis) measured at temperature during the annealing / extension step (64°C in this example). This signal is expected to include fluorescence generated by both the TaqMan probe label (cleaved from the probe) and the EF probe label (incorporated into the double-stranded amplicon). The FAM signal in graph 502 of Figure 5A may include composite fluorescence signal data, as described above with reference to Figure 4.

[0065] Graph 504 in FIG. 5A shows the change in FAM fluorescence signal (ΔRn) after baseline detrending (vs. cycle number measured at temperature during the denaturation step, in this example 95°C) and correction by a linear function correlating measurements of the TaqMan probe at 95°C to measurements at 64°C. This signal represents the approximate fluorescence associated with the TaqMan probe at 95°C and is expected to include fluorescence generated by the TaqMan probe label but not significant fluorescence from the EF probe label. The FAM fluorescence signal data measured at 95°C and linearly transformed to provide the signal data shown in graph 504 in FIG. 5A may comprise "first fluorescence signal data" as described above with reference to FIG. 4. Thus, the transformed fluorescence signal data shown in graph 504 in FIG. 5A can be considered "transformed first fluorescence data." As described above, the function used to correlate measurements at 95°C to measurements at 64°C can be determined by comparing TaqMan probe signals measured according to different temperature conditions (e.g., in the same spectral channel, in the absence of spectral similarity of EF or other probe types).

[0066] In the example of FIG. 5A , composite fluorescent signal data (e.g., graph 502 of FIG. 5A ) is measured according to a second set of reaction conditions (i.e., at the annealing or extension step temperature (or 64°C for a combined annealing / extension step)) and captures fluorescent signals from both probe types (i.e., the TaqMan and EF probes). The converted first fluorescent signal data (e.g., graph 504 of FIG. 5A ) approximates the fluorescent signal from one of the probe types (i.e., the TaqMan probe type) according to the first set of reaction conditions (i.e., at the denaturation step temperature of 95°C). As described above, the composite fluorescent signal data and the converted first fluorescent signal data can be used to determine the fluorescent signal from the other probe type (the EF probe type) according to a second set of reaction conditions (i.e., at the annealing or extension temperature of 64°C). The composite fluorescent signal data may be corrected 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., a "given probe type" as discussed herein).

[0067] Graph 506 in FIG. 5A shows the resolved fluorescent signal determined by subtracting the signal in graph 504 (i.e., the converted first fluorescent signal) from the signal in graph 502 (i.e., the composite fluorescent signal). This signal is expected to estimate the fluorescence generated by the EF label separately from the fluorescence due to 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 fluorescent data or the converted fluorescent signal data can be used to quantify / analyze different targets associated with the TaqMan probe in the reaction mixture.

[0068] Thus, the disclosed technology can advantageously enable assaying of multiple targets / analytes in the same reaction mixture through the same amplification process using probes with detectable labels (e.g., fluorophores) that have spectral similarity, e.g., are detectable in the same detection channel (e.g., in the same fluorescence channel), thereby advantageously enabling increased multiplexity of multiplex assays without relying on additional dyes.

[0069] The graphs in Figure 5B represent the same type of signal measurement as Figure 5A, except that in the assay generating the data in Figure 5B, the EF probe was labeled with FAM dye and the TaqMan probe was labeled with VIC dye. Graph 508 shows the fluorescent signal generated by the EF label (FAM), and graph 510 shows the fluorescent signal generated by the TaqMan probe label (VIC). Graph 512 shows a slight change in fluorescence generated by the EF label at the denaturation temperature (i.e., the EF label fluorescence remains substantially at its baseline signal level), while graph 514 shows the fluorescent signal generated by the TaqMan probe label, which is substantially equal (after application of a linear function) to the signal measured at the annealing / extension temperature. Because the TaqMan and EF probes were differentially labeled in this assay, graph 516 shows the resolved signal for the EF label that essentially matches the EF signal at the annealing / extension temperature (in graph 508), and graph 518 shows the expected baseline signal for the TaqMan probe resulting from subtracting the TaqMan label signal during denaturation from the TaqMan label signal during annealing / extension. Thus, the EF-associated fluorescent signal in graph 516 very closely represented a direct measure of the signal from the EF label (e.g., in graph 508).

[0070] Figure 5C compares the derived EF-associated fluorescent signal 520 (graph 506 in Figure 5A) with the measured EF-associated fluorescent signal 522 (graph 516 in Figure 5B), which represents a direct measure of EF label fluorescence, when resolved from the assay of Figure 5A. The results showed a close correlation between the resolved and measured signals. Thus, the results demonstrated that fluorescent signals resulting from different probe types using the same label in the same detection channel can be resolved separately.

[0071] For further reference, Figure 5D shows the fluorescence signal measured over the number of cycles using the TaqMan probe and EF probe compositions during the annealing / extension step (in this example, at a temperature of 65°C) and during the denaturation step (in this example, at a temperature of 95°C).

[0072] Figure 6 shows the results of another assay test involving five different detection channels / dyes, four of which had the same label (dye) in each channel, with different TaqMan and EF probes across the four channels. One channel had only TaqMan probes. The results demonstrate that the fluorescent signals of different probe types can be determined independently, and thus, 9-plex reactions can be effectively performed.

[0073] In view of the foregoing, in some implementations, the disclosed embodiments may include acquiring different luminescence (e.g., fluorescent) signals at different temperatures, times (e.g., reaction stages of thermal cycling), or other conditions of an amplification process (e.g., a PCR process) involving multiple different probe types. The different probe types may include different luminescence signal (e.g., fluorescent) responses at different temperatures or times. The differential luminescence signal responses of the different probe types at different temperatures, times, or other conditions can be used to determine the separated luminescence signals of the different probe types. This can therefore enable multiplex PCR assays in which luminescence signals emitting the same or substantially overlapping emission spectra (spectrally similar) can be used to quantify different target analytes, thus, for example, utilizing a common detection channel to detect multiple analytes.

[0074] End-point PCR embodiment The analytical techniques according to various embodiments can also be used, as is well known to those skilled in the art, to perform conventional end-point PCR processes in which samples are subjected to PCR in large quantities (or in larger reaction volumes that do not aim to capture or not capture single DNA molecules using Poisson statistics). In such PCR processes, measurement of signals from two different probe types can be performed at the end-point cycle of PCR and under different reaction conditions (e.g., denaturation conditions and annealing and / or extension conditions described herein), similar to the approach described above. In the case of an end-point PCR process, the detected label signal will follow that outlined in FIG. 2A, and thus the signal will indicate the presence or absence of the first target and the second target, respectively. Thus, referring to FIG. 3B, end-point cycle signals measured under two different reaction conditions can result in different levels of signal, as outlined in cycle N+2 (which is analogous to the end-point cycle). By comparing the different signal levels obtained using the algorithm shown in Figure 2A and further described above, the presence or absence of a first target nucleic acid and a second target nucleic acid can be determined using detectable labels that have spectral similarity and may be detectable, for example, in the same detection channel.

[0075] Thus, the methods for determining the presence or absence of multiple targets using the multiplexing techniques described herein can also be performed using end-point PCR (i.e., using a larger or larger reaction volume that is not sized to rely on Poisson statistics, as is well known to those skilled in the art, and capturing single molecules of target nucleic acid in the reaction volume). The method for end-point PCR applications includes preparing a reaction mixture containing a first probe type (e.g., TaqMan probe) and a second probe type (e.g., EF probe) configured to specifically interact with a first target nucleic acid and a second nucleic acid target, respectively; subjecting the reaction mixture to an amplification reaction (e.g., PCR); measuring the end-point cycle signal of the reaction mixture under a first set of reaction conditions (e.g., denaturing conditions, such as about 95°C); measuring the end-point signal of the reaction volume under a second set of reaction conditions (e.g., annealing / extension conditions, such as about 65°C); and , measuring a first total luminescence (e.g., fluorescence) signal, including any first luminescence (e.g., fluorescence) signal, if present; measuring a second total luminescence (e.g., fluorescence) signal, including any first luminescence (e.g., fluorescence) signal, if present, and any second luminescence (e.g., fluorescence) signal, under a second set of reaction conditions; and determining the presence or absence of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by estimating the first luminescence (e.g., fluorescence) signal and / or the second luminescence (e.g., fluorescence) signal based on the first total luminescence (e.g., fluorescence) signal and the second total luminescence (e.g., fluorescence) signal. In other words, the techniques described above with reference to Figures 5A and 5B can be applied to end-point PCR assays and analysis processes.

[0076] Formation of extendable fluorogenic (EF) probe templates Figure 7A shows the process of using a tailed primer 1022 specific for a nucleic acid target 1024 to form a target template 1014 to which an EF probe 1012 can hybridize. The tailed primer 1022 comprises a tail 1026 and a target-specific portion 1028. Figure 7B shows a more detailed view of the tailed primer 1022 as a forward primer, the target-specific primer 1023 paired with the tailed primer 1022 as a reverse primer, and the EF probe 1012.

[0077] As shown, in the first step, target-specific portion 1028 hybridizes to target 1024. Extension of target-specific portion 1028 forms tailed amplicon 1025. Primer 1023 pairs with tailed primer 1022, allowing 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.

[0078] In the second stage, EF probe 1012 hybridizes to target template 1014, allowing amplification to continue, for example, as shown in FIG. 3A. As shown, EF probe 1012 includes probe tail 1017, which has substantial homology to probe tail 1026 and is therefore complementary to tail complement portion 1027 of target template 1014. Extension of probe 1012 and target template 1014 results in the formation of double-stranded amplicon 1019. Primer 1023, shown paired with tailed primer 122 herein, also functions as primer 1023 paired with EF probe 11012, as shown in FIG. 3A, allowing the formation of double-stranded amplicon 1019.

[0079] As shown in Figure 7B, tail 1026 can form the 5' end of tailed primer 1022. EF probe 1012 can include a stem-loop portion, with stem portions 1010 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 the opposite side of the stem-loop portion, such that label 1016 and quencher 1018 are in close proximity when a stem-loop structure is formed, but are spaced farther apart when EF probe 1012 is constrained to a more linear structure (e.g., incorporated into a double-stranded amplicon).

[0080] In the embodiment shown, label 1016 is located at or near the 5' end of EF probe 1012, and quencher 1018 is located 3' to label 1016. The positions of label 1016 and quencher 1018 may be reversed in other embodiments. Preferably, as shown, the stem-loop portion is positioned 5' to probe tail 1017 such that the stem-loop portion remains at the end of the amplicon resulting from extension of EF probe 1012, thereby reducing the likelihood of disrupting stem-loop structure formation (when single-stranded).

[0081] In addition to or instead of the EF probes described herein, some embodiments may include other labeled oligonucleotides that generate increased fluorescence (compared to when in a single-stranded state) when incorporated into a double-stranded amplicon, such as during the extension and / or annealing stages of the PCR process. For example, LUX™ primers contain an internal fluorophore that is quenched by a hairpin structure located 5' to the fluorophore. Like EF probes, LUX™ primers incorporate into a double-stranded amplicon and fluorescence increases when the hairpin structure becomes linear. Furthermore, any primer or probe described herein may include one or more locked nucleic acids (LNAs), as known in the art.

[0082] In some embodiments, the tailed primer 1022 and the corresponding (non-tailed) primer 1023 are provided at different concentrations. For example, the primer 1023 may be provided at a higher concentration than the tailed primer 1022. For example, the primer 1023 may be provided at a concentration of about 2X (2 times) to about 30X the concentration of the tailed primer 1022, or about 5X to about 25X the concentration of the tailed primer 1022, or about 10X to about 20X the concentration of the tailed primer 1022. Because the primer 1023 can function both (1) to drive the formation of the target template 1014 (as shown in FIG. 7A) and (2) to drive the formation of the complement 1015 of the extended probe amplicon 1013 (as shown in FIG. 3A), providing it at a higher concentration than the corresponding tailed primer 1022 can beneficially balance the reaction and increase overall reaction efficiency.

[0083] In some embodiments, the EF probe 1012 is provided at a concentration different from the concentration of the tailed primer 1022 and / or the concentration of the primer 1023. For example, the EF probe 1012 may be provided at a concentration higher than the concentration of the tailed primer 1022 but lower than the concentration of the primer 1023. In some embodiments, the EF probe 1012 is provided at a concentration about 2X to about 20X the concentration of the tailed primer 1022, or about 3X to about 15X the concentration of the tailed primer 1022. As noted above, providing the primer 1023 at a relatively high concentration can increase overall reaction efficiency. By providing the EF probe 1012 at a concentration higher than the tailed primer 1022, but not necessarily higher than the primer 1023, more relative amplification may be directed toward the EF probe 1012, as opposed to the tailed primer 1022, while the primer 1023 may still function as the primary factor in increasing reaction efficiency.

[0084] In addition to or instead of "universal" EF probes that use probe tail 1017, other embodiments include and / or utilize EF probes that have a target-specific portion rather than probe tail 1017. Such EF probes can hybridize directly to a target template nucleic acid, as shown in FIG. 7A, to generate target template 114 having a tail-complementary portion 1027. In such embodiments, probe tail 1017 of EF probe 1012 is replaced with a target-specific portion that hybridizes directly to target 1024. The process is otherwise substantially the same as that shown in FIG. 3A. That is, after the EF probe is extended, a subsequent round of annealing / extension extends the complementary strand, forming a double-stranded amplicon that separates the fluorophore and quencher, allowing for fluorescent signal generation.

[0085] Further details on labels / dyes Exemplary, non-limiting detectable labels that can be utilized with the embodiments described herein include, for example: 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'-dimethoxy-fluorescein (JOE)), 5-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein and 6-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein (TET), 5-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein and 6-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514)), pyrene (e.g., Cascade Blue, Alexa Fluor 405), Coumarins (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430), cyanine dyes (e.g., Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, and Cy7); Rhodamines (e.g., 110, 123, B, B200, BB, BG, B extra, 5-carboxytetramethylrhodamine and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5-carboxyrhodamine 6G and 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine) and 6-ROX (carboxy-X-rhodamine), sulforhodamine B can C, sulforhodamine G Extra, 5TAMRA and 6TAMRA (6-carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X), Alexa Fluor fluorophores, a broad class that includes numerous dye types such as cyanines (e.g., Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 500, Alexa 514, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 610, Alexa 633, Alexa 635, Alexa 647, Alexa 660, Alexa 676, Alexa 680, Alexa 700, and Alexa 750); FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine), as well as other types of dyes known to those skilled in the art.

[0086] The fluorophore label is coupled to a dark fluorescence quencher (dark The fluorophore label may be conjugated to a quencher such as Fluorescent Quencher (DFQ), Black Hole Quencher (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl sulfonate / carboxylic acid quencher and Dabcel sulfonate / carboxylic acid quencher, and MGB-NFQ quencher. Fluorophore labels may also include, for example, sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0087] More details on amplification Amplification products obtained by using one or more embodiments described herein can be generated, 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. The amplification process described herein can include PCR (see, e.g., U.S. Pat. No. 4,683,202). In various embodiments, the PCR process can be real-time or quantitative PCR (qPCR), end-point PCR, digital PCR (dPCR), or any combination thereof.

[0088] In some embodiments, the amplification process comprises reverse transcriptase PCR (RT-PCR). The disclosed methods can include, for example, subjecting the target nucleic acid to a reverse transcription reaction prior to amplification by PCR. In some embodiments, the amplification process comprises one-step RT-PCR (e.g., in a single vessel or reaction volume) using one or more reverse transcriptases in combination with one or more DNA polymerases.

[0089] Optionally, specific qPCR assay samples can be placed in individual wells of a single array or multiwell plate, such as, for example, a TaqMan Array Card (see, e.g., Thermo Fisher Scientific, Inc., Waltham, Massachusetts; catalog numbers 4346800 and 4342265) or a MicroAmp multiwell (e.g., 96-well, 384-well) reaction plate (see, e.g., Thermo Fisher Scientific, Inc., Waltham, Massachusetts; catalog numbers 4346906, 4366932, 4306737, 4326659, and N8010560). Optionally, different qPCR assay samples present in different wells of the array or plate can be dried or lyophilized in situ prior to use, and the array or plate can be stored or transported. In some embodiments, the concepts described herein can be used for in situ hybridization applications not necessarily related to PCR.

[0090] Other amplification methods, such as loop-mediated isothermal amplification (LAMP), Other isothermal methods, such as ELISA (isotope isothermal amplification), are also contemplated for use with the assay embodiments described herein.

[0091] The components described herein for enabling multiplexing using probes with spectrally similar detectable labels can be provided in the form of a kit along with one or more additional components for enabling the amplification process, such as dNTPs, DNA polymerase, amplification buffers / reagents, master mix components known in the art, and other components known in the art for enabling or supporting nucleic acid amplification.

[0092] Further details on implementation The principles disclosed herein may be implemented in a variety of formats. For example, the various techniques described herein may be implemented as methods including various operations (e.g., operations of flow diagram 400 in FIG. 4 ) to achieve a particular result or benefit. In addition to the various techniques described above in which data is collected at multiple time points during various cycles of an amplification process (e.g., a qPCR assay), data may be collected at an endpoint cycle of the amplification process (e.g., an endpoint PCR assay) and analyzed according to the techniques described herein. Such endpoint data collection techniques may be used, for example, in genotyping applications. The various techniques for data collection and analysis disclosed are not mutually exclusive and may be utilized in combination. In some examples, the techniques described herein are represented by computer-executable instructions that may be stored on one or more hardware storage devices. The computer-executable instructions may be executable by one or more processors to perform (or configure a system to perform) the disclosed techniques. In some embodiments, a system may be configured to transmit the computer-executable instructions to a remote device to configure the remote device to perform the disclosed techniques.

[0093] The various specific temperatures, times, and temperature ramp rates shown in the drawings and described with respect to various embodiments are exemplary only and should not be understood as limiting the scope of the present disclosure and claims. Other specific temperatures, times, and temperature ramp rates can be used and will be understood from the remainder of the disclosure. Furthermore, the different sets of conditions under which detection of a luminescent signal occurs can differ from one another in different cycles. For example, the denaturation temperature can vary over different cycles but still be considered a common set of conditions for making measurements. The same is true for the various annealing and / or extension steps in different cycles.

[0094] A system for implementing the disclosed embodiments may include various components such as, by way of non-limiting example, a processor, storage, sensors, an I / O system, a communication system, and the like.

[0095] A processor may include one or more sets of electronic circuitry, including 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 storage. Storage may include physical system memory, and may be volatile, non-volatile, or some combination thereof. Furthermore, storage may include local storage, remote storage (e.g., accessible via a communications system, etc.), or some combination thereof.

[0096] In some implementations, the processor may comprise or be configurable to execute any combination of software and / or hardware components operable to facilitate processing using machine learning models or other artificial intelligence based structures / architectures. For example, a processor may include and / or utilize hardware components or computer-executable instructions operable to execute functional blocks and / or processing layers organized in the form of, for example, 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 neural network, a generative adversarial network, a liquid state machine, an extreme learning machine, an echo state network, a deep residual network, a Kohonen network, a support vector machine, a neural Turing machine, or the like, as non-limiting examples.

[0097] In some examples, operations performable by the system may rely at least in part on a communications system for receiving information from a separate system or remote system, which may include, for example, a computing device, a sensor, and / or others. The communications system may include any combination of software or hardware components operable to facilitate communication between on-system components / devices and / or with off-system components / devices. For example, the communications system may include ports, buses, or other physical connections for communicating with other devices / components. Additionally or alternatively, the communications system may include systems / components operable to wirelessly communicate with external systems and / or devices through any suitable communications channel, such as, but not limited to, Bluetooth, ultra-wideband, WLAN, infrared communications, etc.

[0098] The system may include or be in communication with sensors. A sensor may include any device for capturing or measuring data representative of a perceptible or detectable phenomenon. By way of non-limiting example, a sensor may include one or more optical sensors / detectors, microphones, thermometers, barometers, magnetometers, accelerometers, gyroscopes, and / or others.

[0099] Additionally, 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, but not limited to, a display, a touchscreen, a mouse, a keyboard, a controller, a speaker, and / or others, as non-limiting examples. For example, the I / O system may include a display system, which may include any number of display panels, optics, a laser scanning display assembly, and / or other components. In view of the present disclosure, it will be understood that sensors may, in some examples, be utilized as an I / O system.

[0100] The disclosed embodiments may comprise or utilize special-purpose or general-purpose computers, including computer hardware, as discussed in more detail below. The disclosed embodiments may also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media may be any available media accessible 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 do not store computer-executable instructions but simply hold them are "transmission media." Thus, by way of example, and not limitation, the present embodiments may comprise at least two distinctly different kinds of computer-readable media: computer storage media and transmission media.

[0101] Computer storage media (also known as "hardware storage") include RAM, ROM, EEPROM, CD-ROM, RAM-based solid state drives ("SSDs"), flash memory, and phase-change memory. This is computer-readable hardware storage such as memory, PCM), or other types of memory, or other optical disk storage, magnetic disk storage, or other magnetic storage devices, or any other medium that can be used to store desired program code means 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.

[0102] A "network" may include one or more data links that enable the transfer of electronic data between computer systems and / or modules and / or other electronic devices. When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless) to a computer, the computer properly views the connection as a transmission medium. Transmission media may be used to carry program code in the form of computer-executable instructions or data structures and may include networks and / or data links accessible by a general-purpose or special-purpose computer. Combinations of the above are also included within the scope of computer-readable media.

[0103] Furthermore, upon reaching the various computer system components, program code means in the form of computer-executable instructions or data structures may be automatically transferred from transmission computer-readable media to physical computer storage media (or vice versa). For example, computer-executable instructions or data structures received over a network or data link may be buffered in RAM within a network interface module (e.g., a "NIC") and eventually transferred to computer system RAM and / or a less volatile computer-readable physical storage medium of the computer system. Thus, it should be understood that computer-readable physical storage media may be included in computer system components that also (or primarily) utilize transmission media.

[0104] Computer-executable instructions comprise, 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. The computer-executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code.

[0105] The disclosed embodiments may include or utilize cloud computing. Cloud models may consist of a variety of characteristics (e.g., on-demand self-service, wide area network access, resource pooling, rapid elasticity, measured service, 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.).

[0106] Those skilled in the art will appreciate that the present invention may be practiced in networked computing environments 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, etc. The present invention may also be practiced in distributed system environments where tasks are performed by multiple computer systems (e.g., local and remote computer systems) that are linked through a network (either by wired data links, wireless data links, or a combination of wired and wireless data links). In a distributed system environment, program modules may be located in local and / or remote memory storage devices.

[0107] Alternatively or additionally, the functions described herein may be performed, at least in part, by one or more hardware logic components. For example, without limitation, exemplary types of hardware logic components that may be used include Field-Programmable Gate Arrays (FPGAs), Program-Specific Integrated Circuits (ASICs), Application-Specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), central processing units (CPUs), graphics processing units (GSMUs), and the like. processing unit (GPU), and / or others.

[0108] 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 may run on one or more computer systems. The different components, modules, engines, and services described herein may be implemented as objects or processors (e.g., as separate threads) running on one or more computer systems.

[0109] In various implementations, a system for analyte detection in a multiplexed 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: detect at least a first probe type, which may include a first label configured to generate a first luminescent signal, and a second probe type, which may include a second label configured to generate a second luminescent signal having spectral characteristics similar to the first luminescent signal, at one or more time points during one or more cycles of the PCR process; obtaining measured luminescence signal data associated with a composite luminescence signal from a first probe type and a second probe type, the first probe type and the second probe type having different thermal and / or temporal characteristics; and determining resolved luminescence signal data associated with each luminescence signal from one or more given probe types 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 luminescence signal data associated with the composite luminescence signal and the thermal and / or temporal characteristics of one or more of the first probe type and the second probe type.

[0110] Additional implementations of the system may also include one or more of the following features, in any combination: the first label is the same as the second label; the first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first luminescent signal and the second luminescent signal are a first fluorescent signal and a second fluorescent signal, respectively; the acquiring occurs during a first set of reaction conditions and a second set of reaction conditions of an endpoint cycle of the PCR process, the first set of reaction conditions and the second set of reaction conditions being 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 different from the first analyte; the determining may occur in real time during the one or more cycles of the PCR process; the acquiring and determining 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 can include an extendable fluorogenic probe. The instructions, when executed by one or more processors, further cause the one or more processors to perform the following process: acquiring additional measured luminescent signal data during one or more cycles of the PCR process associated with a first temperature condition and / or time for one or more cycles, and the measured luminescent signal data associated with the composite luminescent signal associated with a second temperature condition and / or time for one or more cycles, the second temperature condition and / or time being different from the first temperature condition and / or time. The determination can utilize the measured luminescent signal data associated with the composite luminescent signal and the additional measured luminescent signal data as inputs to generate resolved luminescent signal data associated with each luminescent signal from a given probe type.Using the measured luminescence signal data associated with the composite luminescence signal and the additional measured luminescence signal data as input to generate resolved luminescence signal data associated with each luminescence signal from the given probe type may include applying a transform to the additional luminescence signal data to generate transformed luminescence signal data, and modifying the measured luminescence signal data associated with the composite luminescence signal with the transformed luminescence signal data to generate resolved luminescence signal data associated with each luminescence signal from the given probe type. The transformed luminescence signal data may indicate an approximate luminescence associated with the first probe type at a second temperature condition and / or time.

[0111] When executed by one or more processors, the one or more instructions of the system can further cause the one or more processors to perform a process of quantifying a first target associated with a first probe type based at least on additional measured luminescence signal data, and a process of quantifying a second target associated with a second probe type based at least on resolved luminescence signal data associated with each luminescence signal from a given probe type. Quantifying the first target can include determining the concentration of the first target in a sample subjected to a PCR process, and quantifying the second target can include determining the concentration of the second target in a sample subjected to a PCR process. The first temperature condition and / or time does not overlap with the second temperature condition and / or time. The first temperature condition is higher than the second temperature condition. The second temperature condition is in the range of about 45 to about 75°C. The first temperature condition is in the range of about 80 to about 100°C. The second temperature and / or time condition is associated with the annealing stage, extension stage, or combined annealing and extension stage of the PCR process. The first temperature condition and / or time is associated with a denaturation stage of a PCR process. The PCR process can include multiple PCR cycles. The first probe type can be associated with a cumulative luminescence signal that remains substantially stable over multiple stages of the PCR cycles and that increases cumulatively over multiple PCR cycles. The second probe type can be associated with a temporal luminescence signal that fluctuates over multiple stages in each PCR cycle. Implementations of the described technology can include hardware, methods or processes, or computer software on a computer-accessible medium.

[0112] In various implementations, a method for enabling analyte detection in a multiplexed polymerase chain reaction (PCR) process may include: obtaining, at one or more time points during one or more cycles of the PCR process, measured luminescence signal data associated with a combined luminescence signal from at least a first probe type, which may include a first label configured to emit a first luminescence signal, and a second probe type, which may include a second label configured to emit a second luminescence signal having spectral characteristics similar to the first luminescence signal, wherein the first probe type and the second probe type have different thermal and / or temporal characteristics; and determining resolved luminescence signal data associated with each luminescence signal from a given one of the first and second probe types during the one or more cycles of the PCR process based at least in part on the luminescence signal data associated with the combined luminescence signal and the thermal and / or temporal characteristics of one or more of the first and second probe types.

[0113] Additional implementations of such methods may also include one or more of the following features, in any combination: 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 luminescent signal and the second luminescent signal may be a first fluorescent signal and a second fluorescent signal, respectively; the acquiring may occur during a first set of reaction conditions and a second set of reaction conditions of an endpoint cycle of the PCR process, the first set of reaction conditions and the second set of reaction conditions being different from each other; the amplification process may be an endpoint PCR process; the first probe type may have binding affinity for a first analyte, and the second probe type may have binding affinity for a second analyte 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 fluorogenic probe. Non-cleavable probe types can include extendable fluorogenic probes.

[0114] The method may further include acquiring additional measured luminescent signal data during one or more cycles of the PCR process associated with a first temperature condition and / or time for one or more cycles, and the measured luminescent signal data associated with the composite luminescent signal associated with a second temperature condition and / or time for one or more cycles, the second temperature condition and / or time being different from the first temperature condition and / or time. The determining step may utilize the measured luminescent signal data associated with the composite luminescent signal and the additional measured luminescent signal data as inputs for generating resolved luminescent signal data associated with each luminescent signal from the given probe type. Using the measured luminescent signal data associated with the composite luminescent signal and the additional measured luminescent signal data as inputs for generating resolved luminescent signal data associated with each luminescent signal from the given probe type may include applying a transformation to the additional luminescent signal data to generate transformed luminescent signal data, and modifying the measured luminescent signal data associated with the composite luminescent signal with the transformed luminescent signal data to generate resolved luminescent signal data associated with each luminescent signal from the given probe type. The transformed luminescent signal data may indicate an approximate luminescence associated with the first probe type at a second temperature condition and / or time.

[0115] The method may also include any of the actions described as being executable by the instructions above.

[0116] In yet other implementations, a computer-readable medium can store one or more instructions for performing operations of system and / or method implementations, which may further be implemented with one or more of the feature combinations described above.

[0117] It will be understood how any feature or action disclosed herein may be combined with any one or combination of other features and actions disclosed herein. Furthermore, content or features of any one of the drawings may be combined with or used in conjunction with any content or features used in any of the other drawings. In this regard, content disclosed in any one drawing is not mutually exclusive with, but rather may be combined with, the content of any of the other drawings.

[0118] The described embodiments are to be considered in all respects as illustrative and not restrictive, and all changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. 1. A system for analyte detection in a multiplexed polymerase chain reaction (PCR) process, comprising: one or more processors of at least one computing device; a memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to: acquiring, at one or more time points during one or more cycles of the PCR process, measured luminescent signal data relating to a composite luminescent signal from at least a first probe type comprising a first label configured to generate a first luminescent signal and a second probe type comprising a second label configured to generate a second luminescent signal having spectral characteristics similar to the first luminescent signal, wherein the first and second probe types have different thermal and / or temporal characteristics; and determining resolved luminescence signal data associated with each luminescence signal from one or more given probe types among 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 luminescence signal data associated with the composite luminescence signal and thermal and / or temporal characteristics of one or more of the first probe type and the second probe type.

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

3. 10. The system of 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 that is different from the first analyte.

4. The system of any one of claims 1 to 0, wherein said determining is performed in real time during said one or more cycles of said PCR process.

5. The system of 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 fluorogenic probe.

9. The instructions, when executed by the one or more processors, cause the one or more processors to: further performing a process of acquiring additional measured luminescence signal data during one or more cycles of the PCR process associated with a first temperature condition and / or time for the one or more cycles; 9. The system of claim 0, wherein the measured luminescent signal data associated with the composite luminescent signal relates to a second temperature condition and / or time of the one or more cycles, the second temperature condition and / or time being different from the first temperature condition and / or time.

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

11. utilizing the measured luminescence signal data associated with the composite luminescence signal and the additional measured luminescence signal data as inputs to generate the resolved luminescence signal data associated with the respective luminescence signals from the given probe type; applying a transform to the additional luminescence signal data to generate transformed luminescence signal data; and correcting the measured luminescence signal data associated with the composite luminescence signal with the transformed luminescence signal data to generate the decomposed luminescence signal data associated with the respective luminescence signals from the given probe type.

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

13. The one or more instructions, when executed by the one or more processors, cause the one or more processors to: quantitating a first target associated with said first probe type based on at least said additional measured luminescence signal data; The system of claim 11 or claim 12, further comprising a process for quantifying a second target associated with the second probe type based at least on the resolved luminescence signal data associated with each luminescence signal from the given probe type.

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

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

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 conditions are associated with an annealing step, an extension step, or a combined annealing and extension step of the PCR process.

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

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

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

23. 23. The system of claim 21, wherein the second probe type is associated with a temporal luminescent signal that varies over multiple stages in each PCR cycle.

24. 3. The system of claim 1, wherein the first label and the second label are a first fluorophore and a second fluorophore, respectively, and the first luminescent signal and the second luminescent signal are a first fluorescent signal and a second fluorescent signal, respectively.

25. 3. The system of claim 1, wherein the obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an end-point cycle of the PCR process, and the first set of reaction conditions and the second set of reaction conditions are different from each other.

26. The system of claim 1 , wherein the amplification process is an end-point PCR process.

27. 1. A method for enabling analyte detection in a multiplexed polymerase chain reaction (PCR) process, comprising: acquiring, at one or more time points during one or more cycles of the PCR process, measured luminescent signal data relating to a combined luminescent signal from at least a first probe type including a first label configured to emit a first luminescent signal and a second probe type including a second label configured to emit a second luminescent signal having spectral characteristics similar to the first luminescent signal, wherein the first probe type and the second probe type have different thermal and / or temporal characteristics; determining resolved luminescence signal data associated with each luminescence signal from a given probe type 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 luminescence signal data associated with the composite luminescence signal and thermal and / or temporal characteristics of one or more of the first probe type and the second probe type.

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

29. 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 that is different from the first analyte.

30. 30. The method of any one of claims 37 to 29, wherein said determining is performed in real time during said 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. 32. The method of claim 31 , wherein the first probe type comprises a TaqMan probe and the second probe type comprises an extendable fluorogenic probe.

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

34. acquiring additional measured luminescence signal data during one or more cycles of the PCR process associated with a first temperature condition and / or time; 34. The method of any one of claims 35 to 33, wherein the measured luminescence signal data associated with the composite luminescence signal relates to a second temperature condition and / or time, the second temperature condition and / or time being different from the first temperature condition and / or time.

35. 35. The method of claim 34, wherein said determining utilizes said measured luminescent signal data and said additional measured luminescent signal data associated with said composite luminescent signal as inputs for generating said resolved luminescent signal data associated with said respective luminescent signals from said given probe type.

36. utilizing the measured luminescence signal data associated with the composite luminescence signal and the additional measured luminescence signal data as inputs to generate the resolved luminescence signal data associated with each luminescence signal from the given probe type; applying a transform to the additional measured luminescence signal data to generate transformed luminescence signal data; and correcting the measured luminescence signal data associated with the composite luminescence signal with the transformed luminescence signal data to generate the decomposed luminescence signal data associated with each luminescence signal from the given probe type.

37. 37. The method of claim 36, wherein the converted luminescence signal data represents an approximate luminescence associated with the first probe type at the second temperature condition and / or time.

38. quantitating a first target associated with the first probe type based on at least the additional measured luminescence signal data; and 38. The method of claim 36 or claim 37, further comprising quantifying a second target associated with the second probe type based at least on the resolved luminescence signal data associated with the respective luminescence signals from the given probe type.

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

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

41. 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. 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 is associated with an annealing or extension step of the PCR process.

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

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

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

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

48. 29. 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 luminescent signal and the second luminescent signal are a first fluorescent signal and a second fluorescent signal, respectively.

49. 29. The method of any one of claims 27 and 28, wherein said obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an end-point cycle of said PCR process, said first set of reaction conditions and said second set of reaction conditions being different from one another.

50. 29. The method of any one of claims 27 and 28, wherein the amplification process is an end-point 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: a process for obtaining, at one or more time points during one or more cycles of a PCR process, measured luminescent signal data relating to a combined luminescent signal from at least a first probe type including a first label configured to generate a first luminescent signal and a second probe type including a second label configured to generate a second luminescent signal having spectral characteristics similar to the first luminescent signal, wherein the first probe type and the second probe type have different thermal and / or temporal characteristics; determining resolved luminescence signal data associated with each luminescence signal from a given probe type of at least 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 luminescence signal data associated with the composite luminescence signal and thermal and / or temporal characteristics of one or more of the at least the first probe type and the second probe type.

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

53. 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 that is different from the first analyte.

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

55. 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. 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 fluorogenic probe.

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

58. The one or more instructions, when executed by the one or more processors, cause the one or more processors to: further performing a process of acquiring additional measured luminescence signal data during one or more cycles of the PCR process associated with a first temperature condition and / or time; 58. The computer-readable medium of any one of claims 51 to 57, wherein the measured luminescent signal data associated with the composite luminescent signal is associated with a second temperature condition and / or time, the second temperature condition and / or time being different from the first temperature condition and / or time.

59. 59. The computer-readable medium of claim 58, wherein said determining utilizes said measured luminescent signal data and said additional measured luminescent signal data associated with said composite luminescent signal as inputs for generating said resolved luminescent signal data associated with said respective luminescent signals from said given probe type.

60. utilizing the measured luminescence signal data associated with the composite luminescence signal and the additional measured luminescence signal data as inputs to generate the resolved luminescence signal data associated with the respective luminescence signals from the given probe type; applying a transform to the additional measured luminescence signal data to generate transformed luminescence signal data; and correcting the measured luminescence signal data associated with the composite luminescence signal with the transformed luminescence signal data to generate the decomposed luminescence signal data associated with each luminescence signal from the given probe type.

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

62. The one or more instructions, when executed by the one or more processors, cause the one or more processors to: quantitating a first target associated with said first probe type based on at least said additional measured luminescence signal data; 62. The computer-readable medium of claim 60 or claim 61, further comprising: a process for quantifying a second target associated with the second probe type based at least on the resolved luminescence signal data associated with the respective luminescence signals from the given probe type.

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

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

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

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

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

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

72. 60. 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 luminescent signal and the second luminescent signal are a first fluorescent signal and a second fluorescent signal, respectively.

73. 60. The computer-readable medium of any one of claims 58 and 59, wherein said obtaining occurs during a first set of reaction conditions and during a second set of reaction conditions of an end-point cycle of said PCR process, said first set of reaction conditions and said second set of reaction conditions being different from one another.

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

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