Energy-transfer probes for multiplexed nucleic acid amplification assays

Abstract

Systems, including methods and compositions, for performing multiplexed assays. The methods may include (A) providing a mixture that includes (i) a sample containing one or more of a plurality of distinct nucleic acid targets, (ii) reagents sufficient for amplification of the targets, and (iii) an energy-transfer probe specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the energy-transfer probe for each target; and (D) determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample. Each energy-transfer probe may be configured such that an extent of energy transfer on the probe from a respective donor fluorophore to a respective acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores may be used to assess the degree of amplification of the respective target.

Description

ENERGY-TRANSFER PROBES FOR MULTIPLEXED NUCLEIC ACID AMPLIFICATION ASSAYSCross-References to Related Application

[0001] This application is based upon and claims the benefit of the following U.S. provisional patent applications: Serial No. 63 / 570,776, filed March 27, 2024; Serial No. 63 / 658,188, filed June 10, 2024; Serial No. 63 / 674,263, filed July 22, 2024; and Serial No. 63 / 728,066, filed December 4, 2024. This application is further based upon and claims the benefit of the following Chinese patent applications: Serial No. 202411253683.1 , filed September 6, 2024; and Serial No. 202411257391.5, filed September 6, 2024. This application is further based upon and claims the benefit of U.S. Patent Application Serial No. 19 / 071 ,725, filed March 5, 2025. Each of these priority applications is incorporated herein by reference in its entirety for all purposes.Introduction

[0002] Nucleic acids may be amplified, using techniques such as polymerase chain reaction (PCR), for a variety of reasons. For example, nucleic acids may be amplified to increase their copy numbers (e.g., to create enough nucleic acid for sequencing and / or other analysis). They also may be amplified to determine the presence, concentration, and / or type of specific nucleic acids, or nucleic acid sequences, in a sample (e.g., to identify a pathogen or variant thereof). The latter use may be termed quantitative amplification, such as quantitative (or real-time) PCR (qPCR), and may be used to look for one or more nucleic acid “targets.”

[0003] A standard method for quantitatively monitoring the progress of nucleic acid amplification uses fluorescent quencher probes. These are short pieces of singlestranded nucleic acid, containing a fluorophore and a nearby quencher, that specifically bind to a portion of a target. The intact quencher probes are dark (quenched), prior to amplification, because the fluorophore is quenched by the quencher. However, as nucleic acid amplification proceeds, the quencher probe is hydrolyzed (cleaved), separating the fluorophore and quencher, and the fluorophore becomes fluorescent. Each round of amplification at least approximately doubles the amount of cleaved probe and concomitantly at least approximately doubles the amount of fluorescence until eventually the fluorescence is detectable. The creation of detectable fluorescence confirms the presence and / or type of the target. Thenumber of rounds of amplification at which fluorescence becomes detectable can be used to assay the abundance of the target.

[0004] It often is desirable to search for multiple targets at the same time. For example, such “multiplexed” assays may be used to search for and identify potential pathogens in a biological sample. Multiple quencher probes, each specific to a particular target, may be used in multiplexed assays if each type of quencher probe includes a different fluorophore. However, the ability to distinguish different fluorophores is limited because each will be excited and emit fluorescence over a range of potentially overlapping wavelengths. In practice, no more than a few (e.g., about four, five, six, or seven) quencher probes may be used in a given multiplexed assay, meaning that no more than a few targets can be assayed simultaneously. Larger numbers of targets may be assayed by splitting samples into subsamples across multiple sample wells. Each subsample may then be assayed for a different small number of targets, and, in the aggregate, a larger number of targets can be assayed. However, this often requires a preamplification step to avoid a significant loss of sensitivity, which can introduce quantification bias, and which can delay the results while adding cost and complexity. Thus, there is an acute need for multiplexed nucleic acid assays and associated probes that can search for, identify, and quantify larger numbers of targets without necessarily dividing a sample.Summary

[0005] The present disclosure provides systems, including methods and compositions, for performing multiplexed assays. These systems use energy-transfer probes, each labeled with two or more fluorophores, to enable much higher plex assays than possible using conventional probes labeled with a single fluorophore. The probe design may allow modulation of the signal intensity of fluorophores on a given energy-transfer probe and the creation of fluorescence signatures unique to each probe. These signatures may include the levels and / or ratios of fluorescence from the fluorophores on each probe. The systems may include a method of performing a multiplexed assay that comprises (A) providing a mixture that includes (i) a sample having one or more of a plurality of distinct nucleic acid targets, (ii) amplification reagents sufficient for amplification of the targets, and (iii) an energy-transfer probe specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the energy-transfer probe for each target; and (D)determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample. The energy-transfer probes may each include a respective donor fluorophore and a respective acceptor fluorophore. In some cases, the energy-transfer probes may further include a quencher. The donor and acceptor fluorophores on a given energy-transfer probe may have distinguishable excitation and / or emission spectra. Each energy-transfer probe may be configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores may be used to assess the degree of amplification of the respective target. For example, each energy-transfer probe may independently be configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target, or vice versa. In some embodiments, the systems may further include one or more additional nucleic acid targets and a corresponding one or more quencher probes specific to the additional targets, each quencher probe comprising a fluorophore and quencher, where changes in fluorescence of the quencher probes report on amplification of the respective additional targets. Exemplary energy-transfer probe applications may include infectious disease assays (including HPV assays), proximity ligation assays, multi- omics assays (including early cancer detection), syndromic testing (including respiratory syndromic testing, among others), transplant monitoring, and gene expression assays (including cancer stratification), among others.Brief Description of The Drawings

[0006] FIG. 1 shows a standard quencher probe, an associated single-target amplification assay, and expected signals obtained with the assay.

[0007] FIG. 2 shows a first novel (linear) energy-transfer probe, an associated single-target amplification assay, and expected signals obtained with the assay.

[0008] FIG. 3 shows a second novel (molecular beacon) energy-transfer probe and snapshots from an associated single-target amplification assay.

[0009] FIG. 4 shows a third novel (linear ribose-doped) energy-transfer probe and a snapshot from the associated single-target assay.

[0010] FIG. 5 shows a fourth novel (linear) energy-transfer (quencher) probe and snapshots from an associated single-target amplification assay.

[0011] FIG. 6 is a pair of graphs showing (A) absorption and (B) emission spectra for a set of exemplary fluorophores suitable for use in constructing a set of energy-transfer probes (or a mixed set of energy-transfer probes and quencher probes).

[0012] FIG. 7 is a graphical depiction of an exemplary algorithm for correcting assay results obtained using energy-transfer probes. (A) A graph of an exemplary fluorescence signal as a function of time (in cycles) for a quantitative nucleic acid assay using a quencher probe. (B) Correction of the amplification curve for an energytransfer probe by baseline correction. Cleavage of energy-transfer probes generates unwanted signals from individual acceptor and donor molecules.

[0013] FIG. 8 is a set of graphs showing time-dependent fluorescence signal curves obtained in a PCR assay using the method of the present disclosure in the actual use of an energy-transfer probe to detect a given target.

[0014] FIG. 9 is a flowchart summarizing algorithmic steps that may be used to determine the concentration of each probe in a multiplexed assay containing a mixture of energy-transfer probes and quencher probes.

[0015] FIGS. 10A-10E show the results of separate amplification experiments for the quencher probe Atto425-BHQ targeting the target HPV11 and the energytransfer probe Atto425-FAM targeting the target HPV43.

[0016] FIGS. 11A-11 E show the results of experiment groups A, B, and C using energy-transfer probes and / or quencher probes to achieve multiplex targets detection.

[0017] FIG. 12 is a set of graphs showing time-dependent fluorescence signal curves obtained in 22-plex PCR assays using the method of the present disclosure in the actual use of both linear energy-transfer quencher probes and linear quencher probes to detect a variety of different HPV targets.

[0018] FIG. 13 is a set of graphs showing time-dependent fluorescence signal curves showing how the deconvolution algorithm can be adjusted to compensate for known crosstalk.

[0019] FIG. 14 is a set of cross-titration curves for a variety of HPV serotypes in the presence of 50 to 50,000,000 copies per PCR reaction.

[0020] FIG. 15 is a scatter plot of Ct values generated from single-target samples and multi-target samples using the second exemplary 16-plex assay of Example 10. The linear relationship shows that the assay generates the same results whether one or several (up to 11 ) targets simultaneously are present in the sample.

[0021] FIG. 16 shows an alternative linear energy-transfer quencher probe, like the probe in FIG. 5, where the quencher has been moved closer to the donor and acceptor and where the 3’ end has been blocked.Detailed Description

[0022] The present disclosure provides systems, including methods and compositions, for performing multiplexed assays. The systems may include a method of performing a multiplexed assay that comprises (A) providing a mixture that includes (i) a sample containing one or more of a plurality of distinct nucleic acid targets, (ii) amplification reagents sufficient for amplification of the targets, and (iii) an energytransfer probe specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the energy-transfer probe for each target; and (D) determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample. The energy-transfer probes may each include a respective donor fluorophore and a respective acceptor fluorophore. In some cases, the energy-transfer probes may further include a quencher. The donor and acceptor fluorophores on a given energy-transfer probe may have distinguishable excitation spectra and distinguishable emission spectra, allowing them to be separately excited and separately detected (at least in the absence of energy transfer). Each energy-transfer probe may be configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores may be used to assess the degree of amplification of the respective target. For example, each energy-transfer probe may independently be configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target, or vice versa. When energy transfer is favored before amplification, each energy-transfer probe may comprise an oligonucleotide having a sequence complementary to that of a portion of a respective target, where the donor fluorophore and acceptor fluorophore are bound to the oligonucleotide close enough together to undergo energy transfer before amplification, and where the oligonucleotide is cleaved during amplification, separating the donor fluorophore and acceptor fluorophore, and thus stopping energy transfer after amplification. When energy transfer is favored after amplification, each energy-transfer probe may againcomprise an oligonucleotide having a sequence complementary to that of a portion of a respective target, where the donor fluorophore and acceptor fluorophore are bound to the oligonucleotide close enough together to undergo energy transfer, but where a quencher reduces or eliminates energy transfer before amplification, and where the oligonucleotide is cleaved during amplification, leaving the donor fluorophore and acceptor fluorophore together, but separating the quencher fluorophore, and thus promoting energy transfer after amplification. In some embodiments, the systems may further include one or more additional nucleic acid targets and a corresponding one or more quencher probes specific to the additional targets, each quencher probe comprising a fluorophore and quencher, where changes in fluorescence of the quencher probes report on amplification of the respective additional targets. For example, the fluorophore on the quencher probe may exhibit reduced fluorescence before amplification, when the quencher is held in proximity to the fluorophore, and enhanced fluorescence after amplification (which occurs in the presence of target), when the quencher is separated from the fluorophore. Fluorescence, and the extent of fluorescence energy transfer and / or quenching, may be assayed using any suitable measure, which typically will be fluorescence intensity, but which could also be fluorescence lifetime and / or fluorescence anisotropy, among others. The systems also may include kits and instruments, among others. Further aspects of the present disclosure are described in the following sections: (I) definitions, (II) overview, (III) signal analysis, (IV) examples, (V) advantages and benefits, and (VI) conclusion.I. Definitions

[0023] Technical terms used in this disclosure have meanings that are commonly recognized by those skilled in the art. However, the following terms may be further defined or understood as follows.

[0024] An “amplicon” is a product of an amplification reaction (e.g., a PCR product). Copies of an amplicon may be generated by amplification of a target sequence, such that the amplicon corresponds to the target sequence (i.e., matches the target sequence and / or is complementary to the target sequence). However, the sequence of the amplicon, especially at primer binding sites, may not exactly match and / or may not be perfectly complementary to the target sequence.

[0025] “Amplification” is a process whereby multiple copies are made of an amplicon matching, complementary to, and / or otherwise corresponding to a targetsequence. The process interchangeably may be called an amplification reaction. Amplification may generate a geometric or exponential increase in the number of copies as amplification proceeds (e.g., 1 , 2, 4, 8, 16, 32, ... 2n, for n cycles). Typical amplifications may produce a greater than 100-fold, 1 ,000-fold, 10,000-fold, 100,000- fold, or million-fold increase, among others, in the number of copies of an amplicon. Exemplary amplification reactions for the probes and methods disclosed herein may include a polymerase chain reaction (PCR) or a ligase chain reaction (LCR), each of which is driven by thermal cycling. The methods also or alternatively may use other amplification reactions, which may be performed isothermally, such as branched- probe DNA assays, cascade-RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid based amplification (NASBA), nicking enzyme amplification reaction (NEAR), PAN-AC, Q-beta replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, stranddisplacement amplification, and / or the like. Amplification may utilize a linear or circular template.

[0026] “Amplification reagents” are any reagents that promote generation of an amplicon by amplification of a target sequence. The reagents may include any combination of at least one primer, primer pair, or more for amplification of at least one target sequence, at least one polymerase enzyme and / or ligase enzyme (which may be heat-stable), and nucleoside triphosphates (dNTPs and / or NTPs), among others.

[0027] “And / or” is used to mean all combinations of the listed elements. For example, a list with two elements “A and / or B” covers three possibilities: only A, only B, or both (A and B). Similarly, a list with three elements “A, B, and / or C” covers seven possibilities: only A, only B, only C, both A and B, both A and C, both B and C, or all three (A, B, and C). The extension to four or more elements follows the same pattern.

[0028] “Complementary” means related by the rules of base pairing. A first nucleic acid polymer, or region thereof, is “complementary” to a second nucleic acid polymer if the first nucleic acid polymer or region is capable of hybridizing with the second nucleic acid polymer in an antiparallel fashion by forming a consecutive (uninterrupted) or nearly consecutive series of base pairs (e.g., at least 5, 6, 7, 8, 9, or 10 consecutive base pairs). The first nucleic acid polymer (or region thereof) is termed “perfectly complementary” to the second nucleic acid polymer if hybridization of the first nucleic acid (or region thereof) to the second nucleic acid polymer forms a consecutive series of base pairs using every nucleotide of the first nucleic acid polymeror region thereof. A “complement” of a first nucleic acid polymer or region thereof is a second nucleic acid polymer or region thereof that is perfectly complementary to the first nucleic acid polymer or region thereof. The “complementarity” between a first nucleic acid polymer (or region thereof) and a second nucleic acid polymer (or region thereof) refers to the number or percentage of base pairs that can be formed when the first nucleic acid polymer (or region thereof) is optimally aligned for hybridization in an antiparallel fashion with the second nucleic acid polymer (or region thereof). Here, “antiparallel” means that, when hybridized, one nucleic acid is in a 5’ to 3’ orientation, while the other nucleic acid is in a 3’ to 5’ orientation (i.e., a 5’ carbon end of one nucleic acid polymer is closer to a 3’ carbon end of the other nucleic acid polymer, and vice versa). A first nucleic acid polymer or region thereof that is complementary to a second nucleic acid polymer or region thereof generally has a complementarity of at least 80%, 90%, 95%, or 100%.

[0029] “Comprising,” “including,” and “having” (and conjugations thereof) are used interchangeably to mean including but not necessarily limited to, and are open- ended terms not intended to exclude additional, unrecited elements or method steps.

[0030] “Crosstalk” refers to an unwanted and / or unintended transfer or bleed over of assay signal between the discrete detection channels used in PCR instruments. The transfer is typically partial, meaning that some or most of the signal remains in the intended or expected channel, but some of the signal also is detected in another channel or channels. PCR instruments standardly use a particular excitation channel and particular emission channel for each fluorophore, where the excitation channel uses excitation light having a preselected range of wavelengths to excite the fluorophore, and the emission channel detects emission light over a preselected range of (typically red-shifted) wavelengths. However, if the construction of a probe and / or the conditions of an assay cause the excitation and / or emission spectra for a fluorophore to change, the associated excitation and emission channels may also (at least partially) change. These changes can complicate assay design and analysis. For example, if emission bleeds into a second (unexpected) channel, then detecting fluorescence only in the standard channel may underreport signal and thus amplification. Such issues can be addressed, if known, by changing fluorophores and / or the deconvolution algorithm, among other approaches; see Example 10.

[0031] A “digital assay” is an investigative procedure(s) capable of detecting single copies of an analyte, such as a nucleic acid target, in a set of subsamples orpartitions, in which each subsample / partition of only a subset of the subsamples / partitions contains one or more copies of the analyte. A “digital amplification assay” is a digital assay that utilizes an amplification reaction(s) to facilitate detection of single copies of a target(s). A digital assay may be performed with any suitable number of subsamples / partitions that gives a statistically significant result, such as at least twenty, one hundred, one thousand, or ten thousand, among others. The subsamples or partitions may comprise spatially isolated volumes, such as aqueous droplets in an immiscible carrier fluid, such as oil, and / or contents of distinct wells in a multiwell plate, among others. The partitions may have any suitable volume(s) for the assay, typically less than about 1 pL. Data analysis may include counting partitions positive for specific targets and using a statistical analysis, such as a Poisson statistical analysis, to determine a concentration of those targets based on ratios of the positive partitions to a total number of partitions (e.g., a sum of the numbers of positive and negative partitions). Here, “positive” means that a partition contains (or appears to contain) at least one copy of the specific target of interest, and “negative” means that a partition does not (or does not appear to) contain the specific target. Digital assays may be especially useful for detecting rare mutations, quantifying genetic and copy number variations, and low abundance (e.g., trace) DNA, among others.

[0032] “Energy transfer,” as used herein, is any non-radiative transfer of energy from a first fluorophore (a “donor fluorophore” or “donor”) to a distinct second fluorophore (an “acceptor fluorophore” or “acceptor”). Energy transfer may include and / or alternatively be termed Forster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET), and / or electronic energy transfer (EET), among others. A consequence of energy transfer is that excitation of a donor may lead to emission from a respective acceptor. Energy transfer is exquisitely sensitive to the separation between donor and acceptor, among other factors, and only occurs when donor and acceptor are very close (typically within about 10 nm, and more typically within about 5-8 nm). Energy transfer, despite being non-radiative, typically requires some overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor.

[0033] “Exemplary” means “illustrative” or “serving as an example.” Similarly, the term “exemplify” (or “exemplified”) means “to illustrate by giving an example.” Neither term implies desirability or superiority.

[0034] “First,” “second,” “A,” “B,” and similar terms are used to distinguish or identify various members of a group (such as “targets” or “fluorophores”), or the like, in the order in which they are introduced in a particular context and are not intended to show serial or numerical limitation.

[0035] “Fluorescence” is optical radiation emitted in response to absorption of light. Fluorescence, as used herein, is intended to cover any form of photoluminescence, in which absorption of one or more photons promotes an electron to an excited state and leads to subsequent emission of a new photon, whether from a singlet state, a triplet state, or other state. Fluorescence may alternatively be referred to as “emission,” depending on context. This is particularly true when “excitation” and “emission” are discussed together. Similarly, “absorption” may alternatively be referred to as “excitation.”

[0036] A “fluorophore,” also termed a “dye,” is any atom, functional group, moiety, or substance capable of fluorescence (where “fluorescence” is defined above). The fluorophore may be bound or otherwise associated with an oligonucleotide, or other portion of a respective probe, using any suitable, but stable, method. Typically, a fluorophore will be covalently bound to an oligonucleotide. However, in some cases, the fluorophore may be associated non-covalently (e.g., by intercalation, hydrogen bonding, electrostatic interaction, encapsulation, etc.).

[0037] An “HPV” is a human papillomavirus (HPV), the most common sexually transmitted infection (STI) in the United States.

[0038] “Light” means electromagnetic radiation in the optical spectrum, namely, ultraviolet light, visible light, and / or infrared light. The term “optical radiation” may alternatively be used in place of “light.”

[0039] The term “nucleic acid” means one or more nucleic acid polymers. A “nucleic acid polymer” is a molecule or molecular duplex of any length composed of naturally occurring nucleotides (e.g., where the polymer is an RNA polymer (also called RNA) or a DNA polymer (also called DNA)), or a compound produced synthetically that can hybridize with DNA or RNA in a sequence-specific manner analogous to that of two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. A nucleic acid polymer may be composedof any suitable number of nucleotides, such as at least about 5, 10, 100, or 1000, among others.

[0040] A “nucleic acid polymer” may have a natural or artificial structure, or a combination thereof. Nucleic acid polymers with a natural structure, namely, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have a backbone of alternating pentose sugar groups and phosphate groups. Each pentose group is linked to a nucleobase (e.g., a purine (such as adenine (A) or guanine (G)) or a pyrimidine (such as cytosine (C), thymine (T), or uracil (U))). Nucleic acid polymers with an artificial structure are analogs of natural nucleic acids and may, for example, be created by changes to the pentose and / or phosphate groups of the natural backbone and / or to one or more nucleobases. Exemplary artificial or otherwise unusual nucleic acid polymers include glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), left-helical nucleic acids (L-DNAs), locked nucleic acids (LNAs), threose nucleic acids (TNAs), xeno nucleic acids (XNA), Z-nucleic acids (Z-DNAs and Z- RNAs), and the like.

[0041] The sequence of a nucleic acid polymer is defined by the order in which nucleobases are arranged along the backbone. This sequence generally determines the ability of the nucleic acid polymer to hybridize with another nucleic acid by hydrogen bonding. In particular, adenine pairs with thymine (or uracil), and guanine pairs with cytosine.

[0042] An “oligonucleotide” is a relatively short and / or chemically synthesized nucleic acid polymer. The length of an oligonucleotide may, for example, be 5 to 1000 nucleotides, among others. Oligonucleotides may function as “primers” (see definition below). Alternatively, or in addition, oligonucleotides may comprise the backbone of quencher probes, energy-transfer probes, and control probes. Oligonucleotides used in probes may be labeled with at least one fluorophore (quencher probes, energytransfer probes, and control probes), at least two fluorophores (energy-transfer probes), and / or at least one quencher (quencher probes, control probes, and some energy-transfer probes). These fluorophores and quenchers may be conjugated to any suitable structure of the oligonucleotide and at any suitable position, including a 5’- end, a 3’-end, or intermediate the 5’- and 3’-ends. In quencher probes, typically the fluorophore(s) will be conjugated to the 5’-end, and the quencher(s) will be conjugated to the 3’-end. In energy-transfer probes, typically the donor fluorophore(s) will beconjugated to the 5’-end, and the acceptor fluorophore(s) will be conjugated to the 3’- end. The oligonucleotide may or may not have secondary structure, such as hairpins.

[0043] A “primer” is an oligonucleotide (DNA / RNA or an analog thereof) capable of serving as a point of initiation of template-directed nucleic acid synthesis or ligation under appropriate reaction conditions (e.g., in the presence of a template to which the primer anneals, nucleoside triphosphates, and an agent for polymerization (such as a DNA or RNA polymerase or ligase, or a reverse transcriptase), in an appropriate buffer and at a suitable temperature). The primer may have any suitable length, such as 5 to 500 nucleotides, among others. The primer may be a member of a “primer pair” or “primer set” including a “forward primer” and a “reverse primer” that define the ends of an amplicon generated in an amplification reaction. (The adjectives “forward” and “reverse” are arbitrary designations relative to one another.) The forward primer hybridizes with a complement of the 5’-end region of a template sequence to be amplified, and the reverse primer hybridizes with the 3’-end region of the template sequence. The term “primer binding site” refers to a portion of a template (or its complement) to which a primer anneals. The full sequence of the primer need not be perfectly complementary to the primer binding site, just sufficiently complementary to anneal under the conditions of the reaction. Accordingly, the primer may have a 3’- end region that is complementary to the primer binding site, and a 5’-end region that is not complementary to the primer binding site (and forms a “5’-tail”). The primer may be a target-specific primer and / or a universal forward primer, among others.

[0044] A “probe,” or “amplification probe,” is a construct configured to enable detection of the occurrence of an amplification reaction and / or formation of an amplicon by the amplification reaction. The amplification probe may be a fluorescent probe including an oligonucleotide labeled with a fluorophore and either a quencher (“quencher probe,” “control probe,” and some “energy-transfer probes”) or second fluorophore (“energy-transfer probe”). (In some embodiments, quencher and control probes may include more than one fluorophore (e.g., to increase signal).) The amplification probe may be configured to hybridize with at least a portion of an amplicon generated by amplification. The amplification probe may be mostly or exclusively linear, such as with a simple (e.g., TaqMan®) hydrolysis probe, or may, at least at times, include stem-loop hairpins or other secondary structure, such as with a molecular beacons probe. The probe may or may not be hydrolyzed during amplification assays, depending on assay design. The terms “quencher probe,”“control probe,” and “energy-transfer probe” import the additional meanings and characteristics described elsewhere in the present disclosure.

[0045] A “quencher” is any atom, functional group, moiety, or substance capable of reducing or eliminating (i.e., “quenching”) the fluorescence, or fluorescence emission, of a fluorophore. A given quencher may work with a single fluorophore, a class of fluorophores, or all fluorophores used in a particular assay. In other words, in an assay with fluorophores A and B, the respective quenchers QA and QB may be the same or different. The ability of quenchers described in the present disclosure to quench fluorescence is distance dependent, such that a given quencher only (significantly) “quenches” fluorescence from nearby fluorophores, specifically, fluorophores bound to or otherwise associated with the same probe. Quenchers may function via any suitable quenching mechanism, including Forster resonance energy transfer (to a dark (i.e., nonfluorescent) acceptor), Dexter electron transfer, exciplex formation, static quenching, and / or collisional quenching, among others. Quenchers may be bound or otherwise associated with oligonucleotides, or other portions of a probe, or probe complement, via the same or similar mechanisms used to bind or otherwise associate fluorophores and probes (see the definition of “fluorophore” above). Quenchers are typically “dark,” meaning that they are a light sink and not themselves luminescent. However, in some cases, the quenchers may be luminescent, as long as they quench luminescence from the fluorophore(s) they are intended to quench and the quencher luminescence cannot be confused with fluorophore luminescence.

[0046] A “sample” is a compound, composition, and / or mixture of interest, from any suitable source(s), directly or indirectly suitable for multiplexed PCR analysis. Samples may be analyzed in their natural state, as collected, and / or in an altered state, for example, following storage, preservation, extraction, lysis, dilution, concentration, purification, filtration, mixing with one or more reagents, pre-amplification, partitioning, or any combination thereof, among others. Clinical samples may include blood, plasma, cell-free plasma, buffy coat, saliva, urine, semen, secretions, stool, sputum, mucous, wound swab, milk, a fluid aspirate (e.g., spinal tap, amniotic fluid, ascites, etc.), a swab (e.g., a nasopharyngeal swab), a wash (e.g., a nasopharyngeal wash, a bronchoalveolar lavage (BAL), etc.), and / or tissue (e.g., a tissue biopsy), among others. Environmental samples may include water, soil, aerosol, and / or air, among others. Research samples may include cultured cells, primary cells, bacteria, viruses,spores, small organisms, any of the clinical samples listed above, or the like. Additional samples may include foodstuffs, weapons components, biodefense samples to be tested for bio-threat agents, suspected contaminants, and so on. Samples may be collected for any suitable purpose, including diagnostics, testing, monitoring, and so on.

[0047] A “subsample” is a smaller sample provided by a bulk sample or samplecontaining fluid and containing only a portion of the bulk sample or sample-containing fluid. A set of subsamples may be a set of partitions, and vice versa. Subsamples may be used to increase multiplexing, for example, by dividing a sample into subsamples and assaying different targets, or sets of targets, in each subsample. Alternatively, or in addition, subsamples may be used in digital assays.

[0048] “Substantially” means to be predominantly conforming to the particular dimension, range, shape, concept, or other aspect modified by the term, such that a feature or component need not conform exactly, so long as it is suitable for its intended purpose or function. For example, a “substantially cylindrical” object means that the object resembles a cylinder but may have one or more deviations from a true cylinder (such as a slightly elliptical versus purely horizontal right cross-section).

[0049] A “target” (also called a “target sequence”) is a nucleic acid polymer sequence (DNA and / or RNA) of any suitable length that is amplified in an amplification reaction. Exemplary target sequences may be about 20-1000 nucleotides, or about 50-500 nucleotides, among others.

[0050] A “template” is a nucleic acid polymer (e.g., an RNA or DNA polymer) that serves as a pattern for the generation of another nucleic acid polymer.II. Overview

[0051] This section provides a brief overview of standard quencher-probe assays, as a counterpoint, and the novel energy-transfer probe assays and mixed energy-transfer probe and quencher probe assays provided by the present disclosure.

[0052] FIG. 1 shows a standard quencher probe, an associated single-target amplification assay, and expected snapshots of exemplary fluorescence signals obtained with the assay. The quencher probe comprises a short oligonucleotide or other backbone capable of specifically binding to a nucleic acid target of interest, a fluorophore, and a quencher. The fluorophore and quencher are bound to the oligonucleotide and sufficiently close together that the quencher “quenches”fluorescence from the fluorophore. In other words, the quencher at least substantially prevents the fluorophore from fluorescing when illuminated by otherwise suitable fluorescence-excitation light, whether the probe is free in solution or bound, prior to amplification, to a target. Amplification of the target leads to the hydrolysis, or cleavage, of the oligonucleotide portion of the quencher probe and separation of the fluorophore and quencher. The separated fluorophore is capable of fluorescence. Thus, with each round of amplification and associated degradation of quencher probe, the amount of free (unquenched) fluorophore rises, which is reflected in an increase in fluorescence signal.

[0053] FIG. 2 shows a first exemplary novel energy-transfer probe, an associated single-target amplification assay, and expected snapshots of exemplary fluorescence signals obtained with the assay, in accordance with aspects of the present disclosure. The energy-transfer probe, which may be termed a linear hydrolysis probe, comprises a short oligonucleotide or other backbone capable of specifically binding to a nucleic acid target of interest, a donor fluorophore (“donor”), and an acceptor fluorophore (“acceptor”). In short, instead of a fluorophore and quencher, the energy-transfer probe includes two distinct fluorophores having a special relationship with one another. Specifically, the energy-transfer probe is constructed so that at least some of the excitation energy absorbed by the donor fluorophore will be transferred non-radiatively to the acceptor fluorophore. Thus, prior to amplification, excitation of the donor will lead to at least some emission from the acceptor, whether the probe is free in solution or bound to the target. In other words, exciting the donor fluorophore will lead to emission from both the donor fluorophore and the acceptor fluorophore, with the proportion of fluorescence from each depending on the efficiency of energy transfer for a given donor and acceptor pair under the conditions of the assay (e.g., the distance between the donor and acceptor in the respective energy-transfer probe). However, after amplification and the associated cleavage of the energy-transfer probe, excitation of the donor will lead only to emission from the donor (or to an increased amount of fluorescence from the donor and a decreased amount of fluorescence from the acceptor if not all of the energy-transfer probe has been consumed in the assay). Thus, with each round of amplification and associated degradation of energy-transfer probe, the amount of free donor rises, which is reflected in an increase in donor fluorescence and a decrease in acceptor fluorescence (at least when the donor is being excited). In essence, the energy-transfer probes effectively create new dyes from existing dyes, because their absorption and emission spectra before and after cleavage differ from those of the constituent dyes alone.

[0054] FIG. 3 shows a second exemplary novel energy-transfer probe and associated assay configurations. The energy-transfer probe, which may be termed a molecular beacons probe, comprises an oligonucleotide (or other suitable backbone), a donor fluorophore, and an acceptor fluorophore. The oligonucleotide in the molecular beacon probe (FIG. 3) may be longer than the oligonucleotide in the linear hydrolysis probe (FIG. 2). However, the associated donor and acceptor in the molecular beacon probe may be as close, or closer, to one another than in the linear hydrolysis probe due to secondary structure in the molecular beacons probe. Specifically, the molecular beacons probe may include a hairpin loop in which portions of the oligonucleotide adjacent the two ends of the probe bind one another and the middle portion of the probe, which contains the target-binding sequence, forms a loop. Thus, if donor and acceptor are positioned at or near the two ends of the oligonucleotide, they may be very close together (for example, bound to separate bases in a complementary base pair, among other possibilities). The molecular beacons probe may undergo energy transfer before amplification when donor and acceptor are nearby. However, after amplification, the donor and acceptor may be much farther apart, leading to a reduction in energy transfer if the probe has not been cleaved, or they may be separated, leading to a significant or complete drop in energy transfer, if the probe has been cleaved during amplification. In this regard, the molecular beacon probe would create signals similar to the signals from the linear hydrolysis probe. A molecular beacons quencher probe may be created by replacing one of the fluorophores with a suitable quencher.

[0055] FIG.4 shows a third exemplary novel energy-transfer probe and associated assay configurations. The energy-transfer probe, which may be termed a doped linear energy-transfer probe, comprises an oligonucleotide (or other suitable backbone), a donor fluorophore, and an acceptor fluorophore. Here, one ribose base (represented by R* in FIG. 4), or more, is doped into the oligonucleotide sequence, while other bases are still deoxyribose bases. The ribose base can be selected from A (adenine), G (guanine), C (cytosine), or U (uracil), among others. The ribose base is preferably located near or in the middle position of the oligonucleotide sequence. Assays using this energy-transfer probe also may include primers, RNase H2 enzyme,polymerase (e.g., Taq DNA polymerase), nucleoside triphosphate (dNTP and / or NTP), target template, and buffer. Manganese (Mn) may be added to the buffer to replace the commonly used magnesium (Mg). When the energy-transfer probe is paired with the corresponding target template sequence (e.g., during annealing), the ribose base can be removed by the RNase H2 enzyme (if present), cleaving the energy-transfer probe. Then, both denaturation and extension steps can separate the donor and acceptor fluorophores on the energy-transfer probe, resulting in a decrease in energy transfer from the donor fluorophore to the acceptor fluorophore. Specifically, when the energy-transfer probe pairs with the target template, RNase H2 enzyme binds to the ribose base in the oligonucleotide sequence and cleaves it, thus releasing the ribose base. At this moment, although the energy-transfer probe breaks due to the lack of one base, there is still energy transfer between the donor and accepter fluorophores due to the bridging of the energy-transfer probe by the target template sequence. In the subsequent extension steps, under the action of Taq DNA polymerase, the primer will be extended. When the primer is extended to the binding site between the energytransfer probe and the target template, the energy-transfer probe will be completely cleaved, resulting in a substantial or complete decrease in energy transfer between the donor and acceptor fluorophores. Optionally, in the subsequent denaturation steps, the energy-transfer probe and the target template sequence can be denatured and separated by heating up. At this time, without the bridging of the target template, the donor and acceptor fluorophores will be completely separated due to the cleavage of the energy-transfer probe, resulting in a substantial or complete decrease in energy transfer between the donor and acceptor.

[0056] FIG. 5 shows a fourth exemplary novel energy-transfer probe, associated assay configurations, and expected snapshots of exemplary fluorescence signals obtained with the assay. The energy-transfer probe, which may be termed a linear energy-transfer quencher probe, comprises an oligonucleotide (or other suitable backbone), a donor fluorophore, an acceptor fluorophore, and a quencher. The donor, acceptor, and quencher are all bound to the oligonucleotide, sufficiently close together that energy transfer can occur from the donor to the acceptor in the absence of the quencher but be disfavored (reduced or eliminated) in the presence of the quencher. Typically, the donor and acceptor are preferentially bound to a portion of the oligonucleotide that is not cleaved (or generally not cleaved) during amplification, allowing the donor and acceptor to stay together after amplification. For example, theymay be bound to portions of the oligonucleotide, such as a 5’ end, that do not bind target. The donor (D) and acceptor (A) may be bound in any suitable order (DA or AD). Donor and acceptor may be separated by a few bases, such as 5 or 10 bases, among others, and separated from the target-binding portion of the oligonucleotide by a few more bases, such as about 5 bases, among others. These spacer portions may have any suitable compositions, including poly-dT, among others. The quencher, in contrast to the donor and acceptor, is preferentially bound to a portion of the oligonucleotide that is cleaved during amplification. For example, the quencher may be bound to a portion of the oligonucleotide that binds to the target. Alternatively, it may be bound to an opposite end, such as a 3’ end, of the oligonucleotide than the donor and acceptor, with cleavage during amplification occurring between the two ends. The quencher may quench fluorescence from the donor and / or acceptor, depending on assay design, but typically will be chosen preferentially to quench or reduce acceptor fluorescence. The oligonucleotide may have any suitable length, for example, about 27-45 bases (9-15 nm length), among others, with target-binding sequences of about 22-31 bases, among others. The separation between the donor and acceptor pair and the quencher, and thus the efficiency of quenching, may be influenced by oligonucleotide length, especially if the donor and acceptor pair and the quencher are positioned on opposite ends of the oligonucleotide. In general, decreasing the separation will increase quenching efficiency and thereby increase the dynamic range of the assay. Some assays involve a calibration fluorophore, which may be matched to a tail of the probe(s).

[0057] A significant advantage of energy-transfer probes over quencher probes is their ability to achieve greater multiplexing with a given number of fluorophores. The number of quencher probes that can be created from a set of fluorophores is equal to the number of fluorophores in the set. However, the number of energy-transfer probes that can be created from the same set of fluorophores may be much larger due to combinatorics. See Example 1. Specifically, sets of 3, 4, 5, 6, 7, and 8 distinct fluorophores can only be used to create 3, 4, 5, 6, 7, and 8 distinct quencher probes, respectively. However, sets of 3, 4, 5, 6, 7, and 8 distinct fluorophores can be used to create up to 3, 6, 10, 15, 21 , and 28 distinct energy-transfer probes, respectively. Thus, while energy-transfer probes can be used to assay for any number of targets, including small numbers of targets, their use becomes particularly advantageous when there are more than a few targets (especially more than about four, five, six, or seventargets). For example, while seven fluorophores can only be used to assay seven targets with quencher probes, seven fluorophores can be used to assay up to 21 targets with energy-transfer probes.

[0058] FIG. 6 shows (A) absorption and (B) emission spectra for a set of exemplary fluorophores suitable for use in constructing a set of energy-transfer probes (or a mixed set of energy-transfer probes and quencher probes). The “red shift” between absorption and emission for a given fluorophore is clearly visible as a rightward (longer-wavelength) shift in the associated absorption and emission spectra for that fluorophore. The red shift between pairs of candidate donor and acceptor fluorophores is visible as a rightward shift in absorption spectra and a corresponding rightward shift in emission spectra for different fluorophore pairs.

[0059] A quantitative nucleic acid amplification system, such as a qPCR system, may include discrete channels for fluorophore excitation and emission. These may be set up for standard dyes and / or dye sets. For example, if a standard quencherprobe assay is based around 4 dyes, namely, dyes A, B, C, and D, then the system may have four excitation channels, one for each of the four dyes, and four detection channels, again one for each of the four dyes. The standard assay would involve exciting in Excitation Channel A while detecting in Emission Channel A, which would reveal fluorescence from dye A, exciting in Excitation Channel B while detecting in Emission Channel B, which would reveal fluorescence from dye B, and so on. However, this approach will change for an energy-transfer probe. For example, with an energy-transfer probe having a donor A and an acceptor B, an assay could involve exciting in Excitation Channel A and detecting in Emission Channel B (to detect energy-transfer only from dye A to dye B). Alternatively, with the same donor and acceptor pair, an assay could involve exciting in Excitation Channel A and detecting in Emission Channels A and B (to detect both direct donor emission from dye A and indirect energy-transfer-mediated acceptor emission from dye B). An exemplary energy-transfer probe can be constructed using FAM as a donor and ROX as an acceptor. In this case, the probe can be excited using a FAM Excitation Channel and detected using a ROX Emission Channel or using both a FAM Emission Channel and a ROX Emission Channel. More generally, especially in systems with multiple fluorophores, in which each fluorophore may take on multiple roles (e.g., as the fluorophore in a quencher probe, as a donor in one or more distinct energy-transfer probes, and / or as an acceptor in one or more distinct energy-transfer probes),emission may be measured across multiple channels and / or measured continuously or quasi-continuously across wavelengths, for example, using a spectrometer instead of discrete channels. In each case, signals from different channels or spectra can be mathematically deconvolved to determine the original concentration of each dye and thus the associated target in the sample.

[0060] Energy-transfer probe assays thus differ from quencher probe assays in significant ways. For example, in standard quencher-probe assays, fluorescence from a quencher probe increases monotonically with increasing nucleic acid amplification. However, in an energy-transfer probe assay, the fluorescence from acceptor and donor fluorophores may rise or fall with amplification, depending on probe and assay design. For example, with energy-transfer probes lacking a quencher (e.g., as shown in FIGS. 2-4), the extent of energy transfer may decrease with amplification (because donor and acceptor are separated), meaning donor fluorescence will increase and acceptor fluorescence will decrease with amplification when the donor is excited. In contrast, with energy-transfer probes having a quencher (e.g., as shown in FIG. 5), the extent of energy transfer may increase with amplification (because the donor and acceptor pair are separated from the quencher), meaning acceptor fluorescence will increase with amplification when the donor is excited. In mixed probe assays, which include combinations of energy-transfer probes and quencher probes, the signals can be even more complex. In summary, signals from different fluorophores in energytransfer probe assays and mixed probes assays are convolved, such that the results of these assays are not inherently obvious.

[0061] Significantly, despite producing more complex signals, energy-transfer probe assays and mixed energy-transfer probe and quencher probe assays can be performed using existing amplification instruments. More specifically, the assays can be performed using the same 3, 4, 5, 6, 7, or 8 excitation and emission channels used with quencher-probe assays, even though they may include as many as 3, 6, 10, 15, 21 , and 28 probes, respectively, each with a unique optical signature determined by the particular combination of donor and acceptor fluorophore. See Example 4. The signals obtained by exciting each fluorophore in the system and measuring associated emission from each fluorophore in the system (or the subset of fluorophores expected to emit when exciting a given fluorophore) can be used to ascertain which probes have been degraded and thus what targets are present. Moreover, the time-evolution of the signal can be used to ascertain the concentrations, or relative concentrations, of thetargets. Methods for deconvolving signals and determining target concentrations are described in the next section.III. Signal Analysis

[0062] This section describes exemplary algorithms for deconvolving the fluorescence signal in amplification assays employing energy-transfer probes or mixed sets of energy-transfer probes and quencher probes.111. A. Probe Deconvolution

[0063] The presence of multiple fluorophores, and the interplay among them, complicates the analysis of fluorescence signals generated during multiplexed assays involving energy-transfer probes. This is particularly true in multiplexed assays in which the number of targets exceeds the number of fluorophores. In such cases, as described above, a given fluorophore may do double duty as both a donor and an acceptor. See Examples 1 and 2. Moreover, the same fluorophore may be used in more than one type of probe, for example, a quencher probe and one or more energytransfer probes. See Example 2.

[0064] The complexity this generates can be illustrated with a simple example. Consider a fluorophore that is (A) the fluorophore in a quencher probe for a target 1 , (b) the donor in an energy-transfer probe for a target 2, and (C) the acceptor in an energy-transfer probe for a target 3. In a multiplexed assay in which the fluorophore is illuminated with suitable excitation light in the channel to detect target 1 , fluorescence from the fluorophore will (A) increase if target 1 is present (because cleavage of the associated quencher probe during amplification will uncouple the fluorophore and quencher, allowing the fluorophore to fluoresce), (B) increase if target 2 is present (because cleavage of the associated energy-transfer probe will uncouple the donor and acceptor, allowing the fluorophore in its capacity as a donor to fluoresce or fluoresce more strongly (because it will no longer transfer energy to the acceptor)), and (C) decrease if target 3 is present (because cleavage of the associated energytransfer probe will uncouple the donor and acceptor, causing the fluorophore in its capacity as an acceptor to fluoresce more weakly or stop fluorescing (because it will no longer receive energy transferred from the donor) in the channel to detect target 3; and remain constant whether target 3 is present or not, in the channel to detect target 1. In this lattermost case, if the acceptor is directly illuminated with suitable excitationlight, its fluorescence will remain at least substantially constant whether or not it is capable of receiving energy transfer from the donor. Moreover, the timing and magnitude of these changes may vary with respective target concentrations.

[0065] Despite this complexity, the amplification curves for every target in a sample can be recovered mathematically, via software, without ambiguity for multiple targets.

[0066] Specifically, the solution may be obtained by performing successive deconvolution operations, beginning with color deconvolution. Color deconvolution mathematically calculates the concentration of each dye (including energy-transfer probes) in the sample. During PCR, as an energy-transfer probe is hydrolyzed, the concentration of cleaved probe can be measured by the system by subtraction of the signal from a baseline, as shown in FIG. 7, generating a normal “qPCR” curve for the energy-transfer probe. The calculations make use of the fact that the ratio Energy- Transfer Probe: Acceptor Dye: Donor Dye = 1 :1 :1 , by chemical design. In this figure, the concentration of energy-transfer probe is shaped like an inverted qPCR curve, and the donor and acceptor dye concentrations increase by exactly the same amount as the decrease in energy-transfer probe.

[0067] FIG. 8 shows time-dependent (number of amplification cycles) fluorescence signal curves obtained based on the disclosed method in a PCR assay of a given target using an energy-transfer probe. The donor fluorophore on the energytransfer probe is Atto425, and the acceptor fluorophore is Cy5. The energy-transfer probe is in the form of a molecular beacon probe (see, e.g., FIG. 3). As PCR proceeds, the deconvolution signal of fluorescence from the energy-transfer probe gradually decreases (as shown in FIG. 8A, that is, the degree of energy transfer from the donor fluorophore Atto425 to the acceptor fluorophore Cy5 gradually decreases. After subtracting the fluorescence deconvolution signal of the energy-transfer probe from baseline through the system algorithm, a normal positive PCR curve can be obtained (as shown in FIG. 7B), which can represent the number (or concentration) of cleaved energy-transfer probes over time (number of amplification cycles). At the same time, the dye concentrations of Atto425 and Cy5 released due to the cleavage of the energytransfer probe increased. The corresponding baseline corrected fluorescence curves of Atto425 and Cy5 are shown in FIGS. 8C and 8D, respectively.

[0068] While it is possible to recover the amplification curves for the energytransfer probes, the individual dyes will be detected in the other channels, which couldbe interpreted as amplification for another assay with the same cycle threshold, Ct (i.e., the number of cycles or time for producing statistically measurable fluorescence). To deconvolve these signals, a matrix can be used to relate the individual dyes generated by the cleavage of a FRET probe to the concentration of the cleaved probe. This can be expressed mathematically:[Concentration of Donor Dye] = [(Cleaved Probe)FRET] (1a)[Concentration of Acceptor Dye] = [(Cleaved Probe)FRET] (1 b)

[0069] Equations (1a) and (1 b) may be generalized for all energy-transfer probes in an assay to determine the concentration of dye generated from the cleavage of these probes. To illustrate this, consider an assay with 3 energy-transfer probes (dyes AB, dyes BC, and dyes AC):Cleaved DyeDye A and 1 Dye B and 0 Dye CCleaved DyeDye A and 1 Dye B and 1 Dye CCleaved DyeDye A and 0 Dye B and 1 Dye C (2)

[0070] In matrix form, the concentration of dye, De, generated by the cleavage of the energy-transfer probes may be expressed asDc= TC' (3)

[0071] whereDye A generated from FRET dye cleavageDe = Dye B generated from FRET dye cleavage (4) Dye C generated from FRET dye cleavage

[0072] Once Dc is calculated from Equation (3), its values may be subtracted from the measured individual dye concentrations to determine the concentration ofdye that has not been generated from energy-transfer probes (i.e., from the quencher probes, if present):DQuencher-Probe Assays—Divieasured — De (7)

[0073] whereDye A generated from Quencher-Probe Assay DQuencher-Probe Assays = Dye B generated from Quencher-Probe Assay (8)Dye C generated from Quencher-Probe Assay

[0074] andDye A calculated from Dye DeconvolutionDMeasured—Dye B calculated from Dye Deconvolution (9) Dye C calculated from Dye DeconvolutionIII.B. Determination of the Transformation Matrix, T

[0075] The elements of the transformation matrix, T, are determined by the expected stoichiometry of the probes (and not by calibration). For the energy-transfer probes proposed, this stoichiometry will be either 1 or 0, depending. Other values are possible using other FRET configurations (e.g., 2 donor dyes and 1 acceptor dye). See Example 4.IILC. Full Correction of Signals

[0076] FIG. 9 is a flowchart summarizing algorithmic steps that may be used to determine the concentrations of the individual dyes and energy-transfer probe dyes in a multiplexed assay containing a mixture of quencher probes and energy-transfer probe.IV. Examples

[0077] The following examples present further aspects of the present disclosure, including energy-transfer probes, mixtures of energy-transfer probes and quencher probes, and associated assays. The examples are intended for illustration and should not be interpreted as limiting the entire scope of the present disclosure. Each example may include one or more distinct embodiments, and / or contextual or related information, function, and / or structure.Example 1 - Probe Combinatorics

[0078] This example describes aspects of probe combinatorics, specifically, the numbers and types of energy-transfer probes that can be constructed using a given number of distinct fluorophores; see Table II. Fluorophores are denoted using alphabetical designators: A, B, C, D, E, F, G, and H.Table I. Exemplary energy-transfer probes that can be constructed using up to eight unique fluorophores.

[0079] The maximum possible number, N, of unique energy-transfer probes that can be constructed from n distinct fluorophores is given by the following equation:N = n(n — l) / 2

[0080] This maximum number assumes that all combinations of fluorophores are capable of energy transfer. In practice, the number may be smaller because energy transfer may only occur with certain probe combinations. Assume that thegeneral direction of energy transfer is A -> B -> C -> D -> E -> F -> G. In other words, assume that the excitation and emission spectra of each successive dye — A, B, C, D, E, F, and G — is red-shifted relative to the excitation spectra of all preceding dyes. The extent of this red shift increases with increasing dye separation in the list (e.g., the red shift between A and D is greater than the red shift between A and C which, in turn, is greater than the red shift between A and B). For this reason, for a given set of fluorophores, one fluorophore can only be a donor (the lowest indexed letter in each group, e.g., A in a group consisting of A, B, C, and D), and one fluorophore can only be an acceptor (the highest indexed letter in each group, e.g., C in a group consisting of A, B, and C; D in a group consisting of A, B, C, and D; and so on). The remaining fluorophores may function as donors on some energy-transfer probes and acceptors on other energy-transfer probes. For example, in a set of energy-transfer probes constructed from fluorophores A, B, C, and D, A would only be a donor, D would only be an acceptor, B would be an acceptor in the pair AB and a donor in the pairs BC and BD, and C would be an acceptor in the pairs AC and BC and a donor in the pair CD. In some cases, one fluorophore (or more fluorophores) may be set aside for use on a control probe and not used on any other probes. In this case, the total number of probes that can be constructed from n fluorophores will be N = (n - l)(n - 2) / 2 + 1, where the first term corresponds to the equation above, with each occurrence of n reduced by one, and the second term is the number of control probes (i.e., one). The control probe(s) may report on one or more aspects of amplification independent of the presence or absence of target and therefore may be used to confirm that the instrument and assay are performing properly, to provide one or more correction terms for analyzing and / or interpreting assay results, and so on.Example 2 - Exemplary Mixed Probe Set

[0081] This example describes exemplary sets of mixed PCR probes constructed from seven fluorophores A, B, C, D, E, F, and G; see Table II. These sets may include a combination of quencher probes and energy-transfer probes or, in some cases, only energy-transfer probes. There may be up to 7 quencher probes, each comprising a unique one of the fluorophores (e.g., A or B or C ...) and a suitable quencher (e.g., QA or QB or Qc ..., respectively) capable of measurably quenching fluorescence from the respective fluorophore when the probe is intact. The quenchersmay be the same or different for each fluorophore (i.e., QA = QB or QA QB, etc.). There also may be up to 21 energy-transfer probes, each comprising a unique pair of fluorophores (e.g., A and B, A and C, and so on). Assume, like in Example 1 , that the general direction of energy transfer is A -> B -> C -> D -> E -> F -> G. Here, the energy-transfer probes are constructed using subsets of the fluorophores: six pairs with no intervening fluorophores (i.e., nearest neighbors) (AB, BC, CD DE, EF, and FG), five pairs with one intervening fluorophore (AC, BD, CE, DF, and EG), four pairs with two intervening fluorophores (AD, BE, CF, and DG), three pairs with three intervening fluorophores (AE, BF, and CG), two pairs with four intervening fluorophores (AF and BG), and one pair with five intervening fluorophores (AG). Exemplary choices of fluorophores may include (I) A = FAM, B = HEX, C = TAMRA, D = ROX, E = ATTO 590, F = Cy5, and G = Cy5.5, and (II) A = FAM, B = HEX, C = TAMRA, D = ROX, E = Cy5, F = Cy5.5, and G = DY 750, among many others, where the fluorophores are listed in order of increasing red shift. The number of energytransfer probes, and thus the number of total probes, may be decreased slightly to 18 and 25, respectively, by excluding energy-transfer probes having four and five intervening fluorophores. This would correspond to excluding energy-transfer probes with the largest wavelength shifts between donor emission and acceptor absorption. Such an exclusion could help ensure stronger energy transfer and thus stronger probe signals, especially with probes such as linear probes (as potentially opposed to molecular beacon probes) having a greater physical distance between donor and acceptor.Table II. Exemplary mixed PCR probe set constructed from seven dyes A-G.The mixed set includes up to 28 total probes, including up to 7 quencher probes and up to 21 energy-transfer probes.

[0082] Any suitable subsets of these PCR probes may be used in a given assay. For example, the quencher probes may be omitted, leaving up to 21 energy-transfer probes. Alternatively, or in addition, some of the energy-transfer probes may be omitted. For example, omitting the energy-transfer probes with four and five intervening fluorophores generates a mixed probe set with 25 members, specifically, 7 quencher probes, and 18 energy-transfer probes. Other probe sets may be created by omitting fluorophores, generating smaller sets, or adding fluorophores (e.g., 8 fluorophores, like in Example 1), potentially generating larger sets. In some cases, a fluorophore may be used only once, for example, in connection with a control probe.Example 3 - Multi-Donor, Multi -Acceptor, and Multi-Quencher Probe Sets

[0083] This example describes exemplary sets of energy-transfer probes involving more than one donor, more than one acceptor, more than one quencher, or combinations thereof on each probe. For example, multi-fluorophore probes constructed from fluorophores A and B could include any combination of AAB, ABA, ABB, BAB, AAAB, AABB, ABBB, and so on, where A is a donor, and B is an acceptor. In this case, probes with more than one donor (e.g., AAB, AABB) may be more effective at increasing acceptor fluorescence prior to probe cleavage, because multiple donors may act as multiple antennae for the acceptor, absorbing more excitation light, and transferring more excitation-state energy to the donor. Similarly, probes with more than one acceptor may be more effective at increasing energy transfer and thus decreasing donor fluorescence prior to probe cleavage. Energytransfer quencher probes with more than one quencher may be darker before amplification, reducing baseline fluorescence and / or increasing dynamic range. Energy-transfer probes having more than one donor, more than one acceptor, and / or more than one quencher may be used alone, together with other such energy-transfer probes, together with two-fluorophore (and, optionally, one quencher) energy-transfer probes, and / or together with quencher probes, among others.Example 4 - Exemplary Assay Signals

[0084] This example describes exemplary assay results for a minimally multiplexed assay involving two fluorophores, A and B, used to construct three probes: (1 ) two quencher probes, AQA and BQB, and (2) one energy-transfer probe, AB, where A is the donor and B is the acceptor. The results, taken alone, for the two quencher probes are straightforward: (1 ) excite A and get no fluorescence if Target 1 is absent and fluorescence from A alone if Target 1 is present, and (2) excite B and get no fluorescence if Target 2 is absent and fluorescence from B alone if Target 2 is present. In contrast, the results, again taken alone, for the energy-transfer probe are more complicated. Excite A and get fluorescence from B (or from both A and B) if Target 3 is absent and from A (or more from A and less from B) if Target 3 is present. The results when two or more targets are present will be a sum (“net signal”) of the individual signals from each probe. See Table III.Table III. Exemplary results from an assay using quencher probes AQA and BQB and energy-transfer probe AB for Targets 1 , 2, and 3, respectively, when all three targets are present in a sample. More specifically, the table elements show how the respective signals evolve during amplification, with initial results to the left of the respective arrows and final results to the right of the respective arrows. The “Net Signal,” as defined in the text, is a sum or composite of all three signals. The table shows simplified results that apply when the quenchers are 100% effective in intact probe, the energy transfer is 100% effective in intact probe, and all probe for a given target is consumed during the assay when the target is present.Example 5 - Exemplary Fluorophores

[0085] The PCR probes of the present disclosure, including energy-transfer probes, quencher probes, and control probes, may be constructed using any suitable fluorophores and fluorophore combinations. This means, for a given assay, that the fluorophores should have distinguishable spectra allowing a given fluorophore to be preferentially, although not necessarily exclusively, excited and allowing fluorescence from that fluorophore to be preferentially, although not necessarily exclusively, detected. Preferential excitation may be achieved by limiting the wavelength(s), or wavelength range(s), of the excitation light used to illuminate the sample, for example, by selective use of certain light sources, certain excitation filters, certain dichroic mirrors, and so on. Preferential detection may be achieved by limiting the wavelength(s), or wavelength range(s), that impinge upon the detector, for example, by selective use of certain emission filters, certain dichroic mirrors, certain monochromators, and so on. Fluorophores chosen for energy-transfer probes should be capable of energy transfer from a relatively blue-shifted donor fluorophore to a relatively red-shifted acceptor fluorophore. Typically, as noted elsewhere in the present disclosure, the emission spectrum of the donor fluorophore will overlap with the absorption or excitation spectrum of the acceptor fluorophore. For example, a peak in the donor emission spectrum may be less than or equal to about 25 nm, 50nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, or 200 nm, among others, from a peak in the acceptor absorption spectrum. The extent of spectral overlap may preferably be higher when donor and acceptor are farther apart on the energy-transfer probe, such as with linear energy-transfer probes, and lower when donor and acceptor are closer together on the energy-transfer probe, such as with molecular beacon probes. Donor and acceptor pairs may be selected more generally by determining and / or confirming experimentally that energy transfer occurs under the conditions of the respective assays. Fluorophores may have absorption and emission wavelengths in the ultraviolet, visible, and / or infrared. They may include one-off fluorophores and / or fluorophores that, via modifications, form part of a fluorophore family. Exemplary fluorophores and fluorophore families may include, among others, and without limitation, fluorophores that are derivatives of Coumarin, Acridine, Rhodamine, Carbopyronine, and Oxazine, of which commercially available forms may be known as ATTO, CY, FAM, HEX, JOE, MAX, ROX, TAMRA (also called TMR), TET, and VIC; fluorescein and rhodamine; ATTO 425, ATTO 430LS, ATTO 488, ATTO 490LS, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101 , ATTO 590, ATTO 633, and ATTO 647N; Cy3, Cy5, and Cy5.5; DY 750; SUN; TEX 615; and Tye 563, Tye 665, and Tye 705. The use of large Stokes-shift (LS) fluorophores, such as ATTO 430LS and ATTO 490LS, allows the creation of novel additional detection channels because they can be excited in standard excitation channels and the resulting fluorescence detected in a channel red-shifted by one or more channels from the standard emission channel for the respective excitation channel. Some fluorophores, such as cyanines, may be prone to aggregation, especially at higher concentrations and / or after thermocycling. Such aggregation can adversely affect the associated assays, for example, by changing fluorophore spectra (which can affect the deconvolution algorithm) and / or by reducing the ability of the probe to bind to target. Changes in fluorophore spectra can cause one fluorophore to be confused for another (for example, if the excitation or emission spectra bleed into channels corresponding to other fluorophores). Aggregation and associated complications may be reduced by reducing the concentrations of such fluorophores, and / or substituting other fluorophores.Example 6 - Exemplary Quenchers

[0086] The PCR probes of the present disclosure, including energy-transfer probes, quencher probes, and control probes, may be constructed using any suitable quencher(s). Exemplary quenchers may include black hole quenchers (BHQ0, BHQ1 ,BHQ2, and BHQ3), blackberry quenchers (BBQ), dabcyl quenchers (DAB), eclipse quenchers (Eclip), and QSY quenchers, among others. Quenchers used in exemplary probes and assays described in the present disclosure include black hole quencher 1 (BHQ1 ) and black hole quencher 2 (BHQ2). Certain quenchers may be more effective with certain fluorophores than others. For example, BHQO may be most effective for fluorophores emitting between about 430 and 520 nm, BHQ1 may be most effective for fluorophores emitting between about 480 and 580 nm, BHQ2 may be most effective for fluorophores emitting between about 559 and 670 nm, and BHQ3 may be most effective for fluorophores emitting between about 620 and 730 nm. The quencher used in energy-transfer probes typically, but not necessarily, targets acceptor fluorescence. However, the quencher more generally may quench both donor and acceptor fluorescence, because quencher either will reduce acceptor fluorescence when the probe is intact and allow acceptor fluorescence with the probe is cleaved. A quencher that quenches donor fluorescence will concomitantly reduce acceptor fluorescence because less donor excited-state energy will be transferred to the acceptor.Example 7 - Exemplary Applications 1

[0087] The energy-transfer probes and mixed energy-transfer and quencher probes provided by the present disclosure may be used for any suitable purpose or application in any suitable assay, including but not limited to purposes and assays currently investigated using quencher probes.

[0088] Exemplary purposes may include agriculture; biosafety, bioterrorism, and forensics (e.g., to characterize unknowns, for food safety, etc.); clinical and veterinary diagnostics (e.g., to diagnose and type infectious agents, cancers, genetic disorders, etc.); and microbiology (e.g., to characterize and study viruses, bacteria, and parasitic agents), among others. Exemplary viral targets may include cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), hepatitis viruses (including HAV, HBV, HCV, HDV, and HEV), herpes simplex virus (HSV), human immunodeficiency virus (HIV-AIDS), human papilloma virus (HPV), and / or yellow fever virus (YFV), among others. Exemplary bacterial targets may include Chlamydia trachomatis (chlamydia), Escherichia coli (E. coli), Gardnerella vaginalis, Mycobacterium tuberculosis (tuberculosis), Neisseria gonorrhoeae (gonorrhea), streptococcus (including Groups A-H) (e.g., bacterial pneumonia, endocarditis, erysipelas, meningitis, necrotizing fasciitis, pink eye, sepsis, and strep throat, amongothers), Treponema pallidum (syphilis), and / or Trichomonas vaginalis (trichomoniasis), among others. Exemplary bioterrorism targets may include Bacillus anthracis (anthrax), Francisella tularensis (tularemia), variola major (smallpox), and / or Yersinia pestis (plague), among others.

[0089] Exemplary applications may include, among others, the detection and quantification of (1 ) pathogens in a sample for infectious diseases, (2) alleles in SNPs or other genomic variations for cancers and other disorders, (3) epigenetic changes, including DNA methylation and others, for cancers and other disorders, and (4) alien or synthetic sequences for multiplex decoding of universal barcodes or other.

[0090] Exemplary assays may include quantitative PCR (qPCR) for DNA targets, reverse transcription quantitative PCR (RT-qPCR) for RNA (including mRNA or noncoding RNA (ncRNA)) targets, and digital PCR, among others. Here, ncRNA includes functional RNA molecules that are not translated into proteins but that nevertheless may play roles in cellular processes such as transcription and translation, among others. Examples of ncRNA may include micro RNAs (miRNAs), ribosomal RNAs, transfer RNAs, small nucleolar RNAs (snoRNAs), and small nuclear RNAs (snRNAs), among others. Digital PCR typically involves the measurement of fluorescence signals at the end of amplification rather than during amplification. Thus, to use energy-transfer probes or mixed energy-transfer and quencher probes, fluorescence signals for digital PCR could be measured at the beginning of amplification to determine a starting amount of energy-transfer probe and at the end of amplification to allow the unique determination of all end-point reporter concentrations. Alternatively, starting amounts could be determined based on the number or fraction of empty partitions (e.g., droplets) at the end of the assay. These assays, including qPCR and dPCR, may be used for quantification, genotyping / serotyping, or both.Example 8 - Exemplary Sample Holders and Instrumentation

[0091] The multiplexed assays described in the present disclosure may be performed using any suitable sample holders and any suitable amplification and detection device(s).

[0092] The sample holder generally comprises any substrate or other mechanism for holding samples for amplification and / or fluorescence detection. The sample holder may hold one or more discrete samples at one or more distinct samplesites. In some cases, sample sites may be defined by mechanical barriers, such as walls, for example, forming sample wells. In other cases, sample sites may be defined by chemical barriers, such as hydrophobic regions separating hydrophilic regions, or distinct spatially separated binding sites for nucleic acids, proteins, and / or other materials. The sample sites may be separate fluid volumes or share a common fluid volume. Exemplary sample holders with separate volumes may include multiwell plates, such as PCR plates and microplates, among others. Such plates may have any suitable number of sample wells, such as 96, 384, or 1536 sample wells, among others. Exemplary sample holders with a common fluid volume may include nucleic acid sample chips, among others. The samples themselves may be independent of one another or aliquots or replicates of one another, depending on the analysis. They also may be control or calibration samples. Plates may be partially or totally sealed (e.g., using an adhesive film).

[0093] The amplification and detection device(s) generally comprises any quantitative nucleic acid amplification instrument configured to amplify nucleic acid, excite fluorescence from fluorophores, and detect fluorescence emitted by the fluorophores before, during, and / or after amplification. The system may include one or more light sources, a stage and thermocycler, one or more detectors, and a processor configured to control the light source(s), thermocycler, detector(s), and other system components, if present. The light sources may be configured to produce fluorescence excitation light capable of inducing fluorescence from fluorophores used in the analysis. Exemplary light sources may include lasers and / or light-emitting diodes (LEDs), among others. The detectors may be configured to detect fluorescence emission light emitted by probes used in the analysis. Exemplary detectors may include point detectors, such as photodiodes, and imaging detectors, such as pointdetector arrays and / or cameras, such as CCD or CMOS cameras, among others. The system further may include an optical relay structure configured to direct light, such as fluorescence excitation light, from the light source(s) to the sample(s), and to direct fluorescence emission light from the sample(s) to the detector(s). The optical relay structure may include lenses, mirrors, beamsplitters, spectral filters, and neutral density filters, among others. The system may be capable of exciting fluorophores selectively and detecting fluorescence according to wavelength. This may be accomplished using any suitable mechanisms, including spectral filters, dichroic beamsplitters, color-sensitive detectors, and spectrofluorometers, among others.Example 9 - Exemplary Applications 2: 10-Plex HPV Assay

[0094] This example describes an exemplary 10-plex assay for detecting and identifying human papillomavirus (HPV) variants using a mixture of linear energytransfer probes and linear quencher probes. In this assay, the energy-transfer probes and quencher probes are constructed from the following five fluorophores: FAM, TAMRA, ROX, ATTO425, and Cy5. The quencher probes (5 in total) are FAM-BHQ, TAMRA-BHQ, ROX-BHQ, ATTO425-BHQ, and Cy5-BHQ, where BHQ represents quencher. The energy-transfer probes (5 in total) are FAM-TAMRA, FAM-ROX, TAMRA-Cy5, ROX-Cy5, and ATTO425-FAM, where the fluorophore on the left is the donor and the fluorophore on the right is the acceptor. The probes may be linear probes, in some or all cases including poly dT sequences to increase separations between donors, acceptors, and / or quenchers, as desired.

[0095] The targets (10 in total) in this example are HPV11 , HPV16, HPV26, HPV33, HPV35, HPV42, HPV43, HPV56, HPV58, and HPV59. The targets may include suitable gBIock DNA.Experiment 1— Perform amplification assays using 5 quencher probes and 5 energy-transfer probes for their respective targets separately.

[0096] Experiment 1 consists of amplification assays performed using the five quencher probes and five energy-transfer probes described above in single-plex assays. In other words, each probe is used for its respective target separately. The number of targets in each individual amplification assay is 5000 copies, and each assay is repeated 4 times. Table IV shows the composition of the amplification assay reaction mixture. Table V shows the protocol used to prepare the single-plex probe and primer set used in the amplification assay reaction mixture. Single-plex probe and primer set are prepared in advance. Table VI shows the PCR cycling conditions.Table IV. Reaction mixture of Experiment 1 .Table V. Preparation protocol for the single-plex probe and primer set.Table VI. PCR cycling conditions.

[0097] Experiment 1 demonstrates the efficacy of the probes in single-plex format. Data are shown for both an exemplary quencher probe (e.g., Atto425-BHQ targeting HPV11 ) and an exemplary energy-transfer probe (e.g., Atto425-FAM targeting HPV43); see FIG. 10. FIG. 10A shows a PCR curve for quencher probe Atto425-BHQ in the presence of HPV11. The quencher probe Atto425-BHQ cleaves as PCR progresses, separating Atto425 and BHQ. The Atto425 becomes fluorescent, leading to an increase in fluorescence signal over time. FIG. 10B shows an original PCR curve of energy-transfer probe Atto425-FAM in the presence of HPV43. The fluorescence deconvolution signal of the energy-transfer probe decreases as PCR progresses, reflecting a decrease in the extent of energy transfer from the donor fluorophore Atto425 to the acceptor fluorophore FAM. FIG. 10C shows that subtracting the fluorescence deconvolution signal of the energy-transfer probe from baseline by a systematic algorithm yields a conventional forward PCR curve that can represent the number (concentration) of Atto425-FAM cleavage over time (number of PCR cycles). PCR-based amplification, in the presence of HPV43, causes the cleavage of Atto425- FAM over time (number of PCR cycles); during this process, the dye concentrations of cleaved (separated) Atto425 and FAM increase, corresponding to the baseline- corrected fluorescence curves for Atto425 and FAM shown in FIG. 10D and FIG. 10E, respectively. These results show that the quencher probe Atto425-BHQ can effectively identify and detect its corresponding target HPV11 , and that the energy-transfer probe Atto425-FAM can effectively identify and detect its corresponding target HPV43.Experiment 2 - Validate the possibility to achieve multiplex target assays using energy-transfer probes and / or quencher probes.

[0098] Experiment 2 consists of amplification assays using the five quencher probes and five energy-transfer probes of Experiment 1 in multiplex assays. In other words, multiple probes are used simultaneously for multiple targets in a 10-plex assay format. The experiments are divided into three groups: (A) Group A looks at two energy-transfer probes having a same fluorophore (and investigates whether they interfere): Atto425-FAM and FAM-TAMRA; (B) Group B looks at an energy-transfer probe and a quencher probe having a same fluorophore (and investigates whether they interfere): Atto425-FAM and FAM-BHQ1 ; and (C) Group C looks at two energytransfer probes that do not share any fluorophores (and that thus should not interfere): Atto425-FAM and Cy5-ROX.

[0099] The Group A experiments test the effectiveness of two exemplary energy-transfer probes (ATTO425-FAM and FAM-TAMRA) at identifying and detecting their respective targets when the two energy-transfer probes use a same type of fluorophore (FAM). Two sub-experiments were set up under experiment Group A to verify the effectiveness of the two energy-transfer probes when their respective targets are at the same concentration and different concentrations. The first subexperiment, A_1 , has different concentrations of the two targets (the number of HPV43 corresponding to ATTO425-FAM was 5000 copies, and the number of HPV35 corresponding to FAM-TAMRA was 20000 copies), while the second sub experiment, A_2, has the same concentration of two targets (both targets have 5000 copies). Each sub-experiment is repeated 4 times.

[0100] The Group B experiments test the effectiveness of an exemplary energytransfer probe and an exemplary quencher probe (Atto425-FAM and FAM-BHQ) on their respective targets when the energy-transfer probe and the quencher probe use a same type of fluorophore (FAM). Two sub-experiments were set up under experiment Group B to verify the effectiveness of the energy-transfer probe and the quencher probe when their respective targets are at the same concentration and different concentrations. The first sub-experiment, B_1 , has different concentrations of the two targets (the number of HPV43 corresponding to ATTO425-FAM was 5000 copies, and the number of HPV16 corresponding to FAM-BHQ was 20000 copies), while the second sub experiment, B_2, has the same concentration of two targets (both targets had 5000 copies). Each sub-experiment is repeated 4 times.

[0101] The Group C experiments test the effectiveness of two exemplary energy-transfer probes (Atto425-FAM and ROX-Cy5) on their respective targets when the two energy-transfer probes do not use a same type of fluorophore (in other words, when all fourfluorophores on the two probes are different). Two sub-experiments were set up under experiment Group B to verify the effectiveness of the two energy-transfer probes when their respective targets at the same concentration and different concentrations. The first-sub experiment, C_1 , has different concentrations of the two targets (the number of HPV43 corresponding to ATTO425-FAM was 5000 copies, and the number of HPV56 corresponding to ROX-Cy5 was 20000 copies), while the second sub-experiment, C_2, has the same concentration of two targets (both targets have 5000 copies). Each sub experiment is repeated 4 times.

[0102] The experimental conditions and protocols used in Experiment 2 are summarized in Tables VII-IX. Table VII shows the composition of the amplification assay reaction mixture. Table VIII shows the protocol used to prepare the 10-plex probe and primer set used in the amplification assay reaction mixture. 10-plex probe and primer set are prepared in advance. Table IX shows the PCR cycling conditions.Table VII. Composition of the amplification assay reaction mixture.Table VIII. Preparation rules of the 10-plex probe and primer set.Table IX. PCR cycling conditions.

[0103] The experimental results are shown in FIGS. 11A-11 E, where the signal curves for the fluorophore and the energy-transfer probes have been calibrated.

[0104] FIG. 11A shows the PCR curves from experiment A_1 (the PCR curve from experiment A_2, which is not shown here, is similar to FIG. 10A), where the fluorescence corresponding to the fluorescence curve of the fluorophore FAM is emitted by the fluorophore FAM released after cleavage of Atto425-FAM and FAM-TAMRA, and the fluorescence curve of the fluorophore FAM is baseline corrected. The PCR fluorescence curve of the energy-transfer probe Atto425-FAM is corrected by subtracting its original PCR fluorescence curve from original baseline, and the PCR fluorescence curve of the energy-transfer probe FAM-TAMRA is corrected by subtracting its original PCR fluorescence curve from original baseline. The results are as expected, and the sum of the concentrations of the energy-transfer probes Atto425- FAM and FAM-TAMRA is very close to the total concentration of the fluorophore FAM.

[0105] FIG. 11 B shows the PCR curve from experiment B_1 , where the fluorescence corresponding to the fluorescence curve of the fluorophore FAM is emitted by the fluorophore FAM released after cleavage of Atto425-FAM and FAM- BHQ, and the fluorescence curve of the fluorophore FAM is baseline corrected. The PCR fluorescence curve of the energy-transfer probe Atto425-FAM is corrected by subtracting its original PCR fluorescence curve from original baseline, and the PCR fluorescence curve of quencher probe FAM-BHQ is obtained by subtracting the corrected PCR fluorescence curve of the energy-transfer probe Atto425-FAM from the PCR fluorescence curve of fluorophore FAM. The Ct values of the two probes are close to each other, and the deviation between the Ct values of the two probes is within the acceptable range.

[0106] FIG. 11C shows the PCR curve from experiment B_2, where the fluorescence corresponding to the fluorescence curve of the fluorophore FAM is emitted by the fluorophore FAM released after cleavage of Atto425-FAM and FAM- BHQ, and the fluorescence curve of the fluorophore FAM is baseline corrected. The PCR fluorescence curve of the energy-transfer probe Atto425-FAM is corrected by subtracting its original PCR fluorescence curve from original baseline, and the PCR fluorescence curve of quencher probe FAM-BHQ is obtained by subtracting the corrected PCR fluorescence curve of the energy-transfer probe Atto425-FAM from the PCR fluorescence curve of fluorophore FAM. The Ct values of the two probes are close to each other, and the deviation between the Ct values of the two probes is within the acceptable range.

[0107] FIG. 11 D shows the PCR curve from experiment C_1 , where the fluorescence curves of the fluorophore FAM and fluorophore Cy5 both are baseline corrected, the PCR fluorescence curve of the energy-transfer probe Atto425-FAM was corrected by subtracting its original PCR fluorescence curve from original baseline, and the PCR fluorescence curve of the energy-transfer probe ROX-Cy5 was correctedby subtracting its original PCR fluorescence curve from the original baseline. In this experiment, there is a fourfold difference in the concentrations of the two targets and an approximately two-cycle difference in the Ct values, as expected.

[0108] FIG. 11 E shows the PCR curve from experiment C_2, where both the fluorescence curves of the fluorophore FAM and fluorophore Cy5 are baseline corrected, the PCR fluorescence curve of the energy-transfer probe Atto425-FAM is corrected by subtracting its original PCR fluorescence curve from original baseline, the PCR fluorescence curve of the energy-transfer probe ROX-Cy5 is corrected by subtracting its original PCR fluorescence curve from the original baseline. The Ct values of the two probes are close to each other, and the deviation between the Ct values of the two probes is within the acceptable range.

[0109] In summary, from the above results, it can be seen that experiment groups A, B, and C all showed the expected detection and identification effect of the targets with the probes designed herein. For example, in the mixtures of two interfering linear energy-transfer probes, Atto425-FAM and FAM-TAMRA, curves were recovered as predicted. The heights of the FAM curves in these assays were doubled because FAM was used in both probes. In contrast, the heights of the curves for the unique fluorophores, Atto425 and TAMRA, match the respective probe concentrationsExample 10 - Exemplary Applications 3: 16-Plex and 22-Plex HPV Assays

[0110] This example describes exemplary 16-plex and 22-plex HPV assays for identifying HPV variants using a mixture of linear energy-transfer quencher probes and linear quencher probes (such as the probes in FIGS. 5 and 1 , respectively). Assays may be performed using any suitable PCR instrument, including a six, seven, or eight-channel instrument, among others.Experiment 1 : Exemplary 22-Plex HPV Assay

[0111] The performance of an exemplary 22-plex HPV assay is investigated using samples containing single and multiple targets. The 22-plex assay includes 15 linear energy-transfer quencher probes and 7 linear quencher probes; see Table X.Table X. Composition and performance data for 22-plex HPV assay. The characterization of assay “strength” is based on assay endpoint: strong (endpoint > 40 nM), medium (20 nM < endpoint < 40 nM), and weak (endpoint < 20 nM).

[0112] FIG. 12 shows typical PCR amplification curves using the 22-plex HPV assay with samples containing single targets. There are three replicates per assay. The relative “strength” of the assays varies (see Table X), with some assays stronger (higher signal, lower variation) and other assays weaker (lower signal, noisier baseline). Here, higher signals correspond to greater amplification. Generally, weaker assays involve probes containing Cy5 or TAMRA. In summary, despite some weaker assays, amplification for a single target is consistently observed.

[0113] Table XI summarizes results using the 22-plex HPV assay with samples containing multiple targets. Sixteen assay reaction mixes are prepared usingcombinations of 2, 3, or 4 distinct targets. For testing purposes, “problematic” energytransfer quencher probes are used preferentially, with nine of ten probes containing TAMRA or Cy5. All assays amplify as expected at approximately Ct = 25. The average Ct of all assays over all reaction mixes is 24.62+1.31 (where threshold = 2). Poorer assays (e.g., ROX-Cy5) have a delayed Ct.Table XI. Summary of 22-plex HPV assay results on mixtures of 2, 3, and 4 HPV targets. (*) Five of the assays for 4 targets have 1 non-amplifying assay, all involving Cy5-Cy5.5 probe.

[0114] Crosstalk (see Definitions) is observed with two probes: Atto425-TAMRA from the FAM-TAMRA probe (15%), and TAMRA-Cy5 from the TAMRA-ROX probe (10%). However, if crosstalk is known and consistent across assays, as it is here, the deconvolution matrix can be adjusted to compensate. For example, the FAM-TAMRA probe consistently bled about 10-15% into the Atto425-TAMRA channel in both single- plex and multiple target experiments. Using the appropriately modified spectrum for FAM-TAMRA in the deconvolution matrix almost completely compensates for the crosstalk. See FIG. 13 and discussion below. Analogous modifications can be used to offset crosstalk from other probes, such as TAMRA-ROX.Experiment 2: First Exemplary 16-Plex HPV Assay

[0115] The performance of a first exemplary 16-plex HPV assay is investigated using samples containing single and multiple targets; see Tables XII and XIII and FIG. 14. The first 16-plex assay includes 10 linear energy-transfer quencher probes and 6 linear quencher probes. The probes and targets correspond to 16 well-performing probes from the 22-plex assay. Specifically, the 16-plex assay omits the following six probes present in the 22-plex assay: Cy5-Cy55, ROX-Cy5, TAMRA, TAMRA-Cy5, TET-TAMRA, and TET-Cy5. The remaining 16 probes display relatively high signal- to-noise amplification in the 22-plex assay. Nine of the 16 remaining probes still include either Cy5 or TAMRA (but not both).

[0116] The 16-plex assay amplifies all 16 targets in a single well. Results are summarized in Table XII.Table XII. Summary of performance data for 16-plex HVP assay.PCR efficiency is impacted by target concentration. Specifically, a 10-fold increase in target concentration, from 5000 to 50000 copies per reaction, reduces Ct, the number of cycles to threshold, by about 4 cycles. Crosstalk is observed with some fluorophores, including FAM-TAMRA and Atto425-FAM, including in channels where no amplification is expected. However, the levels of crosstalk, even with amplification, are not that large without bias correction (e.g., 12% and 20% for FAM-TAMRA and Atto425-FAM) and can be reduced to negligible levels with bias correction (e.g., ~5% and -6%, again for FAM-TAMRA and Atto425-FAM).

[0117] FIG. 14 shows exemplary cross titration experiments used to assess dynamic range in the 16-plex assay for four pairs of probes selected to challenge the deconvolution algorithms. The goal is to deduce the range of target concentrations over which the assay correctly reports the presence, type, and relative concentration of targets. Each graph shows cross-titration assay results for samples containing two targets, one increasing in concentration from sample to sample, the other decreasing in concentration from sample to sample. Target concentrations range from 50 to 50,000,000 copies per reaction. Seven of eight assays demonstrate amplification with 50 copies per reaction. Eight of eight assays (100%) demonstrate at least 4 logs of dynamic range (in other words, accuracy over a 10,000-fold change in target concentration). Table XIII shows the linear fits to the data in FIG. 14. The coefficient of determination (R squared) for all assays is greater than or equal to 0.96, meaning the data are well fit by lines.FIG XIII. Linear fits and coefficients of determination for multi-target vs. single target plots in FIG. 14.

[0118] Table XIV shows results for eight probes used in the cross-titration studies testing the reproducibility of Ct at 10-times the limit of detection (LoD) for the 16-plex assay. Here, the coefficient of variation, CV, was less than 5% for 100% of assays (7 / 7) tested. Smaller values of CV reflect greater reproducibility in measured values.Table XIV. Reproducibility of Ct at 10-times limit of detection in 16-plex HPV assay. Here, <Ct> is the average (or mean) value of Ct, SD is the standard deviation in the average value of Ct, and CV is the coefficient of variation, or dispersion, in the values of Ct used to calculate <Ct>, where CV is defined as the ratio of the standard deviation to the average value.Experiment 3: Second Exemplary 16-Plex HPV Assay

[0119] The performance of a second exemplary 16-plex HPV assay is investigated using samples containing single and multiple targets; see Tables XV- XVIII and FIG. 15 The second 16-plex assay includes 11 linear energy-transfer quencher probes and 5 linear quencher probes. All probes are labeled with a uniquecombination of fluorophore(s) and quencher. Table XV lists targets, probe types, probe fluorophore(s), and target concentrations (in copy numbers per well) for the assay.Table XV. Composition and assay conditions for the second exemplary 16-plex assay. Targets with zero copies per well are not tested.The 16-plex assay amplifies all targets present in a particular sample in the same well. In this example, assays are performed in triplicate in a 96-well plate. Tables XVI and XVII show the PCR reaction mix and thermal cycling conditions, respectively, for those assays.Table XVI. PCR reaction mix per well for the second exemplary 16-plex assay.Table XVII. PCR thermal cycling conditions for the second exemplary 16-plex assay.

[0120] Each assay is tested either with a single target with the 16-plex assay set or within a multi-target test with 11 targets present in the same well. The reported Cts in single target or multi-target are shown in Table XVIII and plotted in FIG. 15. One of the 11 assays (HPV 81 using the Cy5-Cy5.5 FRET pair) amplifies as a single target but not in the multi-target configuration. Another of the 11 assays (HPV82 using the TET pair) shows a significantly delayed Ct (over 2 cycles) between the single target and multi-target configuration. However, the results are otherwise robust and in very close agreement for the various targets examined, in single or multi-target tests, over a wide range of target concentrations. In particular, the assay gives the same result whether one target or the same target with other targets simultaneously are present in the sample.Table XVIII. Comparison of the Cts for 11 targets in 16-plex assay run as either a single target or in a multi-target mixAssay Accuracy

[0121] Assays should accurately and reproducibly identify targets to have real- world applicability. Various metrics can be used to assess accuracy. For example, diagnostic accuracy reports on how accurately an assay can determine the presence of absence of a target:Diagnostic Accuracy = 100All = True Positive + True Negative + False Positive + False NegativeThis metric has particular value, as here, where a goal is simply to determine whether a condition, such as a particular HPV infection, exists. Table XIX shows results of experiments to determine the diagnostic accuracy of the 16-plex and 22-plex HPV assays under conditions meant to challenge the associated deconvolution algorithms. Samples contain 2, 3, and 4 types of targets. The diagnostic accuracies were uniformly high, between 97.5% and 100%, despite using weaker assay configurations and the presence of crosstalk, further affirming the soundness of assay design and implementation.Table XIX. Calling accuracy for up to four targets in 22-plex and 16-plex HPV assays.Fluorophore Aggregation and Crosstalk

[0122] This section describes potential complications with multiplex assays, such as fluorophore aggregation and crosstalk, and associated fixes. Crosstalk, as described above, refers to the detection of assay signals in unintended channels. Crosstalk is undesirable because it complicates signal analysis. Some crosstalk may reflect shifts in excitation and emission spectra that occur when fluorophores arecombined with other fluorophores, for example, when they are bound together on an energy-transfer probe or brought into proximity when different probes are combined in a multiplex assay. Some fluorophores may aggregate, especially at the higher concentrations used in multiplex assays, which in turn may lead to shifts in excitation and / or emission spectra. Aggregation may also lead to unstable (e.g., drifting) baselines. Aggregation may increase as a function of heating and cooling cycles, sometimes over 20 cycles or more, and may reduce the effective probe concentration by rendering probe (at least temporarily) unavailable or unreactive. These complications can be reduced or circumvented in various ways, including physical fixes and / or software fixes, among others.

[0123] Physical fixes may include the judicious choice of fluorophores and / or modifications to probe structure. For example, particularly problematic fluorophores (e.g., Cy5 and TAMRA) or combinations of fluorophores (e.g., FAM-TAMRA) can be used sparingly, if at all. Alternatively, or in addition, problematic fluorophores may be replaced with more water-soluble alternatives because aggregation appears to correlate with dye hydrophobicity. Accordingly, TAMRA may be replaced with AF555 or sulfonated Cy3, Cy5 may be replaced with Atto643 or sulfonated Cy5, and Cy5.5 may be replaced with Atto680 or sulfonated Cy5.5, among others. Quenchers also may be moved closer to donors and acceptors to increase quenching efficiency and reduce baselines. Blockers, such as C3 or C6, may be added to the 3’ end to prevent non-specific priming. FIG. 16 shows a modified linear energy-transfer quencher probe in which the quencher has been moved closer to the donor and acceptor, for example, at or near the middle of the probe, and the 3’ end has been blocked, relative to the probe in FIG. 5. Crosstalk may drop from single-plex to multiplex conditions. In some embodiments, the relative positions of donor, D, and acceptor, A, may be reversed.

[0124] Software fixes may address baseline shift, crosstalk, and other quantifiable effects if known or measurable. For example, drifting backgrounds can be identified and subtracted and / or data analysis can be confined to more stable portions of the signal. Crosstalk can be characterized and corrected for in the algorithms. For example, if a known fraction of the fluorescence from a given fluorophore is detected in an unintended channel, the algorithm can be adjusted to put the errant signal back where it belongs by rescaling the signal up in the intended channel and rescaling the signal down in the unintended channel.Example 11 - Selected Aspects

[0125] This section describes additional selected aspects of the present disclosure, presented without limitation as a series of paragraphs, some or all of which may be numerically indexed for clarity and efficiency. Each of these paragraphs can be combined with one or more other paragraphs, and / or with disclosure from elsewhere in this application, in any suitable manner. Some of the paragraphs below expressly refer to and further limit other paragraphs, providing without limitation examples of some of the suitable combinations.A. Methods1 . A method of performing a multiplexed assay, the method comprising (A) providing a mixture including (i) a sample containing one or more of a plurality of distinct nucleic acid targets, (ii) reagents sufficient for amplification of the targets, and (iii) an energy-transfer probe specific to each target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target; (B) amplifying the plurality of targets in the mixture; (C) measuring fluorescence from the energy-transfer probe for each target; and (D) determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.1A0a. The method of paragraph 1 , wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target.1A0b. The method of paragraph 1 , wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is disfavored before amplification of the respective target and favored after amplification of the respective target.1A00. The method of any preceding paragraph, wherein the step of measuring fluorescence from the energy-transfer probes includes measuring the fluorescence multiple times during the step of amplifying to yield time-dependent fluorescence data.1A00a. The method of paragraph 1A00, wherein measuring the fluorescence multiple times corresponds to measuring the fluorescence after increasing rounds of amplification (e.g., after each round of amplification).1 AOOb. The method of paragraph 1 A00 or 1 AOOa, wherein measuring the fluorescence multiple times corresponds to measuring the fluorescence across different emission wavelengths (e.g., using different detection channels).1A1. The method of any of paragraphs 1A00 to 1A00b, wherein the timedependent fluorescence data from each energy-transfer probe includes an increase in fluorescence from the respective donor fluorophore and a decrease in fluorescence from the respective acceptor fluorophore when the corresponding target is present.1A2. The method of any of paragraphs 1A00 to 1A00b, wherein the timedependent fluorescence data from each energy-transfer probe includes a decrease in fluorescence from the respective donor fluorophore and an increase in fluorescence from the respective acceptor fluorophore when the corresponding target is present.1 B. The method of any preceding paragraph, wherein the step of measuring fluorescence includes measuring fluorescence from every fluorophore in the system while preferentially exciting a single fluorophore in the system.1 B1 . The method of paragraph 1 B, wherein the step of measuring fluorescence from every fluorophore in the system is performed serially for each fluorophore.1 B2. The method of paragraph 1 B, wherein the step of measuring fluorescence from every fluorophore in the system is performed in parallel for all fluorophores.1 B3. The method of any of paragraphs 1 B to 1 B2, wherein the step of measuring fluorescence includes generating an emission spectrum for each donor fluorophore and each acceptor fluorophore in the mixture, and wherein the step of determining a quantity includes deconvolving the spectra to assess an extent of cleavage for each probe.1 B3a1. The method of paragraph 1 B, wherein the step of measuring fluorescence is performed by placing spectral filters in the light path to determine the wavelength, or wavelength ranges, of light incident on the camera.1 B3a2. The method of paragraph 1 B, wherein the step of measuring fluorescence is performed using a camera with discrete filter bandwidths that determine the wavelength, or wavelength ranges, of light incident on the camera.1 B3b. The method of paragraph 1 B, wherein the step of measuring fluorescence is performed using a spectrofluorometer.1 B4. The method of any of paragraphs 1 B to 1 B3b, wherein the step of measuring fluorescence includes a generating time-dependent fluorescence intensity curve for each energy-transfer probe in the mixture to assess an extent of cleavage for each probe from a baseline representing the concentration of each energy-transfer probe before amplification.IC. The method of any preceding paragraph, wherein the energy-transfer probes include all pairwise combinations of at least four fluorophores, each pairwise combination reporting on a distinct target.I D. The method of any preceding paragraph, wherein a same type of fluorophore is a donor fluorophore on a first energy-transfer probe specific to a first distinct target and an acceptor fluorophore on a second energy-transfer probe specific to a second distinct target different from the first distinct target.I E. The method of any preceding paragraph, the energy-transfer probes being constructed from at least four distinct fluorophores A, B, C, and D, wherein the energytransfer probes include the following combinations of fluorophores: AB, AC, AD, BC, BD, and CD.1 E1 . The method of paragraph 1 E, wherein the step of measuring fluorescence includes sequentially exciting each of fluorophores A, B, C, and D while measuring fluorescence emission serially or in parallel from all of fluorophores A, B, C, and D.1 E2. The method of any preceding paragraph, the energy-transfer probes being constructed from at least five distinct fluorophores A, B, C, D, and E, wherein the energy-transfer probes are selected from the following combinations of fluorophores: AB, AC, AD, AE, BC, BD, BE, CD, CE, and DE.1 E3. The method of any preceding paragraph, the energy-transfer probes being constructed from at least six distinct fluorophores A, B, C, D, E, and F, wherein the energy-transfer probes are selected from the following combinations of fluorophores: AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, and EF.1 E4. The method of any preceding paragraph, the energy-transfer probes being constructed from at least seven distinct fluorophores A, B, C, D, E, F, and G, whereinthe energy-transfer probes are selected from the following combinations of fluorophores: AB, AC, AD, AE, AF, AG, BC, BD, BE, BF, BG, CD, CE, CF, CG, DE, DF, DG, EF, EG, and FG.1 E5. The method of any of paragraphs 1 E2 to 1 E4, wherein the step of measuring fluorescence includes sequentially exciting each of the fluorophores while measuring fluorescence emission serially or in parallel from all of the fluorophores.1 F. The method of any preceding paragraph, wherein the donor fluorophores and acceptor fluorophores are selected from the group consisting of ATTO 425, FAM, HEX, TAMRA, ROX, Cy5, Cy5.5, and DY 750.1 FX. The method of any preceding paragraph, wherein at least one of the energy-transfer probes further includes a quencher, in addition to a donor and acceptor, wherein the quencher reduces fluorescence from at least one of the donor and acceptor when the probe is intact (i.e., before amplification).1 FX1. The method of paragraph 1 FX, wherein all the energy-transfer probes further include a quencher, in addition to respective donors and acceptors, wherein the quencher reduces fluorescence from the respective donor and acceptor when the probe is intact (e.g., before amplification).1 FX2a. The method of paragraph 1 FX or 1 FX1 , the energy-transfer probes having an oligonucleotide backbone, wherein the donor, acceptor, and quencher are attached to the backbone with the acceptor disposed between the donor and quencher.1 FX2b. The method of paragraph 1 FX or 1 FX1 , the energy-transfer probes having an oligonucleotide backbone, wherein the donor, acceptor, and quencher are attached to the backbone with the donor disposed between the acceptor and quencher.1 FX3a. The method of paragraph 1 FX2a or 1 FX2b, the oligonucleotide backbone having two ends, where the donor and acceptor are disposed at or near one end of the backbone and the quencher is disposed at or near the other end.1 FX3b. The method of paragraph 1 FX2a or 1 FX2b, the oligonucleotide backbone having two ends and a middle, where the donor and acceptor are disposed at or near one end of the backbone and the quencher is disposed at or near the middle.1 FXX. The method of any preceding paragraph, wherein a bias correction is applied during signal analysis to account for crosstalk between channels.1 G. The method of any preceding paragraph, the mixture including an additional nucleic acid target different from each target in the plurality of distinct nucleic acid targets, further comprising (A) providing a quencher probe specific to the additional target, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the additional target; (B) amplifying the additional target in the mixture; (C) measuring fluorescence from the quencher probe; and (D) determining from the measured fluorescence a quantity representative of a level of the additional target.1 G0. The method of paragraph G, the probes being constructed from two fluorophores, A and B, and up to two respective quenchers, QA and QB, wherein the energy-transfer probe is formed from the fluorophore combination AB, and wherein the quencher probe is selected from AQA, BQB, or both.1 G1 . The method of paragraph 1 G, wherein the steps of amplifying the plurality of distinct targets and amplifying the additional target are performed simultaneously.1 G1a. The method of paragraph 1 G1 , wherein the steps of measuring fluorescence from the energy-transfer probe for each distinct target and measuring fluorescence from the quencher probe are performed simultaneously.1 G2. The method of any of paragraphs 1 G to 1 G1a, wherein the quenchable fluorophore is selected from the group of fluorophores used to construct the energytransfer probes.1 GX. The method of paragraph 1 G2, wherein the step of determining from the measured fluorescence a quantity representative of a level of the additional target includes subtracting fluorescence data of the energy-transfer probes from measured fluorescence of each total individual fluorophores that cleaved from quencher probes and / or energy-transfer probes (e.g., using Equation 7).1 G3. The method of any of paragraphs 1 G to 1 G2, wherein the energy-transfer probes are constructed from at least four distinct fluorophores A, B, C, and D, wherein the quencher probe is constructed from fluorophore A and a quencher QA, and wherein the set of energy-transfer probes and quencher probes include the following combinations of fluorophores and quenchers: AB, AC, AD, BC, BD, CD, and AQA.1 G3a. The method of paragraph 1 G3, further comprising providing one or more quencher probes BQB, CQC, and DQD constructed from fluorophores B, C, and D and quenchers QB, Qc, and QD.1 G3a1. The method of paragraph 1 G3a, wherein at least some of quenchers QA, QB, Qc, and QD are the same.1 G4. The method of any of paragraphs 1 G to 1G3, the mixture including further additional nucleic acid targets different from each target in the plurality of distinct targets, further comprising performing the steps of providing, amplifying, measuring, and determining using a number of quencher probes less than or equal to the number of distinct fluorophores used to construct the energy-transfer probes.1 G4a. The method of paragraph 1 G4, wherein the number of quencher probes is at least four.1 G5. The method of any preceding paragraph, wherein the number of energytransfer probes exceeds the number of distinct fluorophores used in their construction.1 G6. The method of any preceding paragraph, wherein there are at least seven fluorophores and at least twenty-one energy-transfer probes constructed from the at least seven fluorophores.1 G6a. The method of paragraph 1 G6, wherein there are seven quencher probes constructed using the at least seven fluorophores and corresponding quenchers capable of quenching fluorescence from the at least seven fluorophores.1 G7. The method of any preceding paragraph, wherein the distinct targets are from distinct infectious agents.1 G8. The method of any preceding paragraph, wherein the amplification reagents include one or more primers specific to each distinct or additional target, nucleotide triphosphates, and at least one polymerase.1 G9. The method of any preceding paragraph, wherein the changes in fluorescence from the respective donor fluorophores and respective acceptor fluorophores are changes in an intensity of fluorescence emitted by each fluorophore.1 G10. The method of any of paragraphs 1 to 1 G9, wherein the level of each target is selected from the group consisting of a presence or absence of each target, a concentration of each target, and a copy number of each target.1 G10a. The method of paragraph 1 G10, wherein the level of each target is a presence or absence of each target.1 G1 Ob. The method of paragraph 1 G10, wherein the level of each target is a concentration of each target.1 G10c. The method of paragraph 1 G10, wherein the level of each target is a copy number of each target.1 G11. The method of any preceding paragraph, the multiplexed assay being a multiplexed qPCR assay, wherein the step of amplifying the plurality of distinct targets includes performing a polymerase chain reaction.1 G12. The method of any preceding paragraph, wherein the plurality of distinct nucleic acid targets and additional nucleic acid targets, if present, are derived from a single sample without dividing the sample and performing separate multiplexed assays for different distinct targets on each divided portion or preamplifying nucleic acid in the sample before forming the mixture.1 H. The method of any preceding paragraph, wherein the step of determining a quantity includes relating the concentration of individual fluorophores generated by the amplification-induced cleavage of each energy-transfer probe to the concentration of the cleaved energy-transfer probe.1 J. The method of any preceding paragraph, wherein the energy-transfer probe includes at least two respective donor fluorophores and a respective acceptor fluorophore, each donor fluorophore capable of energy transfer to the respective acceptor fluorophore.1 J1 . The method of paragraph 1 J, wherein the two donor fluorophores have the same excitation spectra.1 J2. The method of paragraph 1 J, wherein the two donor fluorophores have different excitation spectra.1 K. The method of any preceding paragraph, wherein the energy-transfer probe includes at least two respective acceptor fluorophores, each acceptor fluorophore capable of receiving fluorescence excitation energy from the respective donor fluorophore.1 K1. The method of paragraph 1 K, wherein the two acceptor fluorophores have the same excitation spectra.1 K2. The method of paragraph 1 K, wherein the two acceptor fluorophores have different excitation spectra.I L. The method of any preceding paragraph, wherein the energy-transfer probe includes at least two respective donor fluorophores and at least two respective acceptor fluorophores.I M. The method of any preceding paragraph, where fluorescence emitted by the acceptor fluorophore is red-shifted relative to the fluorescence emitted by the donor fluorophore in the absence of an acceptor fluorophore.1 N1. The method of any preceding paragraph, wherein at least one of the energy-transfer probes and / or quencher probes, when present, is a linear hydrolysis probe.1 N2. The method of any preceding paragraph, wherein at least one of the energy-transfer probes and / or quencher probes, when present, is a molecular beacons probe.1 N2a. The method of any preceding paragraph, wherein at least one of the energy-transfer probes and / or quencher probes, when present, has an oligonucleotide backbone, and wherein at least one of the probes includes a ribose base doped into its backbone.1 N3. The method of any preceding paragraph, wherein a 3’-end of at least one probe is blocked.1 N4. The method of any preceding paragraph, wherein the energy-transfer probes and / or quencher probes, when present, include at least two of the following: a linear hydrolysis probe, a molecular beacon probe, a ribose-doped probe, and a blocked probe.I P. The method of any preceding claim, wherein the nucleic acid targets are selected from the group consisting of deoxyribonucleic acid (DNA) targets and ribonucleic acid (RNA) targets.IQ. The method of any preceding claim, further comprising dividing the mixture for digital PCR analysis prior to the step of amplifying the plurality of targets in the mixture, the targets are present in some but not all the partitions, the targets are amplified in each partition, and the fluorescence from each probe for each target is measured in each partition.1.4. A method of performing a multiplexed digital assay, the method comprising (A) forming a mixture including (i) a sample containing one or more of a plurality of distinct nucleic acid targets, (ii) reagents sufficient for amplification of the targets, and (iii) an energy-transfer probe specific to each target in the first set oftargets, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target; (B) dividing the mixture into a plurality of partitions such that a given target is present in some but not all the partitions; (C) amplifying the targets present in each partition; (D) measuring fluorescence from the energy-transfer probe for each target; and I determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.1.4A. The method of paragraph 1.4, wherein the steps of measuring and determining include assessing whether each partition is positive or negative for each target and assigning a concentration of each target based on a ratio of the number of partitions positive for each target to the total number of partitions.1.4B1.The method of paragraph 1.4 or 1.4A, wherein the steps of forming a mixture and dividing the mixture are performed simultaneously.1.4B2.The method of paragraph 1.4 or 14A, wherein the step of forming a mixture is performed before the step of dividing the mixture.1 ,4C. The method of any of paragraphs 1 .4 to 1 .4B2, further comprising the additional step(s) and / or limitation(s) from any of claims 1 to 1Q.1 .5. A method of performing a multiplexed assay, the method comprising (A) providing a mixture including (i) a sample containing one or more of a plurality of distinct nucleic acid targets including a first set of targets and a second set of targets, (ii) reagents sufficient for amplification of the targets, (iii) an energy-transfer probe specific to each target in the first set of targets, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respectivetarget, and (iv) a quencher probe specific to each target in the second set of targets, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the additional target; (B) amplifying the plurality of targets in the mixture; (C) measuring fluorescence from the energy-transfer probe for each target in the first set of targets and from the quencher probe for each target in the second set of targets; and (D) determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.1 ,5A. The method of paragraph 1 .5, wherein at least one of the energy-transfer probes further includes a quencher.1 ,5B. The method of paragraph 1 .5 or 1 ,5A, further comprising the additional step(s) and / or limitation(s) from any of claims 1 to 1Q.B. Kits2. A kit for performing a multiplexed assay for a plurality of nucleic acid targets, comprising an energy-transfer probe specific to each target, wherein each energytransfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target.2A0a. The kit of paragraph 2, wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target.2A0b. The kit of paragraph 2, wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptorfluorophore is disfavored before amplification of the respective target and favored after amplification of the respective target.2A0b0. The kit of paragraph 2A0b, wherein each energy-transfer probe further includes a quencher, in addition to a donor fluorophore and acceptor fluorophore, wherein the quencher reduces fluorescence from the respective donor and acceptor when the probe is intact (e.g., before amplification).2A1 . The kit of any preceding kit paragraph, further comprising at least one quencher probe specific to an additional target, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quencher probe can be used to assess the degree of amplification of the additional target.2A2. The kit of any preceding kit paragraph, further comprising the probe- related limitations of one or more of the following paragraphs: 1C, 1 D, 1 E, 1 E1 , 1 E2, 1 E3, 1 E4, 1 E5, 1 F, 1 FX, 1 FX1 , 1 FX2a, 1 FX2b, 1 FX3a, 1 FX3b, 1 G, 1 G0, 1G1 , 1 G1a, 1 G2, 1G3, 1 G3a, 1 G3a1 , 1 G4, 1 G4a, 1 G5, 1 G6, 1G6a, 1 J, 1J1 , 1J2, 1 K, 1 K1 , 1 K2, 1 L, 1 M, 1 N1 , 1 N2, 1 N2a, 1 N, 1 N1 , 1 N2, 1 N3, and 1 N4.2B. The kit of any preceding kit paragraph, further comprising amplification reagents.2B1. The kit of paragraph 2B, wherein the amplification reagents include one or more primers specific to each distinct (or additional target, if applicable), nucleotide triphosphates, and at least one polymerase.C. Systems3. A system for performing a multiplexed assay for a plurality of nucleic acid targets, comprising (A)a quantitative nucleic acid amplification instrument configured to amplify nucleic acid, excite fluorescence from fluorophores, and detect fluorescence emitted by the fluorophores before, during, and / or after amplification; (B) reagents sufficient for amplification of the targets, and (C) an energy-transfer probe specific to each target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitationspectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target.3A0a. The system of paragraph 3, wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target.3A0b. The system of paragraph 3, wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is disfavored before amplification of the respective target and favored after amplification of the respective target.3A0b0. The system of paragraph 3A0b, wherein each energy-transfer probe further includes a quencher, in addition to a donor fluorophore and acceptor fluorophore, wherein the quencher reduces fluorescence from the respective donor and acceptor when the probe is intact (e.g., before amplification).3A. The system of any preceding system paragraph, wherein the quantitative nucleic acid amplification instrument is a qPCR or dPCR instrument.3B1. The system of any preceding system paragraph, wherein the quantitative nucleic acid amplification instrument includes emission filters to characterize the spectrum of fluorescence emission from the energy-transfer probes.3B2. The system of any of paragraphs 3 to 3A, wherein the quantitative nucleic acid amplification instrument includes a spectrofluorometer to characterize the spectrum of fluorescence emission from the energy-transfer probes.3C. The system of any of paragraphs 3 to 3B2, wherein the quantitative nucleic acid amplification instrument includes a stage configured to support a multiwell plate or reaction chambers on a chip.3C1 . The system of paragraph 3C, further comprising a multiwell plate or chip.3D. The system of any of paragraphs 3 to 3C1 , further comprising the one or more of the probes of paragraph 2A.V. Advantages and Benefits

[0126] The systems described in the present disclosure may have various advantages and benefits, relative to existing systems and / or standing alone. Exemplary advantages and benefits are described here, without limitation, to illustrate and motivate aspects of the present disclosure. Some or all of these advantages and benefits may be present, to a greater or lesser degree, in any given embodiment.

[0127] The energy-transfer probes described herein allow probes to be constructed, and assays to be performed, for more targets than constituent fluorophores when four or more distinct fluorophores are used. In other words, a small number of fluorophores can be used to detect a larger number of targets. This, in turn, allows assays to be constructed and performed for a substantially larger number of targets than conventional quencher-probe based assays. Moreover, the energytransfer probes may optionally be used with quencher probes to achieve even greater multiplexing. Significantly, the increased multiplexing provided by energy-transfer probes can be accomplished using commercially available fluorophores and instruments, including existing qPCR instruments, and, while requiring new probes, does not require any special changes to the fluorophores or optical detection subsystems of the instruments.

[0128] The associated assays also have advantages and benefits. For example, the assays may be performed with larger target counts without sample splitting, maintaining the same sensitivity as a single-tube assay, while reducing costs and complexity. Alternatively, or in addition, the assays may be performed using a single probe per target, again reducing costs and complexity relative to some other assays. For example, approaches like multicolor combinatorial probe coding require the synthesis of many probes having the same sequence but different fluorophores. Moreover, if two targets are present in a sample at approximately the same concentration, the ability to identify and quantify the targets uniquely using these other approaches may be significantly degraded or impossible. Furthermore, data from assays can be recast in a familiar format, such as that used in standard quencherprobe assays, for interpretation by end users.

[0129] The systems described herein also may reduce or minimize problems inherent in amplification assays. For example, only three oligonucleotides are needed for each assay (left and right primers and a probe), which greatly simplifies the bioinformatics challenge to minimize dimer formation during amplification.VI. Conclusion

[0130] The disclosure set forth above may encompass multiple distinct examples with independent utility. Although each of these has been disclosed in its preferred form(s), the specific examples thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. To the extent that section headings are used within this disclosure, such headings are for organizational purposes only. The subject matter of the disclosure includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the present disclosure.

Claims

WHAT IS CLAIMED:1 . A method of performing a multiplexed assay, the method comprising: providing a mixture including(i) a sample containing one or more of a plurality of distinct nucleic acid targets,(ii) reagents sufficient for amplification of the targets, and(iii) an energy-transfer probe specific to each target, wherein each energytransfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target; amplifying the plurality of targets in the mixture; measuring fluorescence from the energy-transfer probe for each target; and determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.

2. The method of claim 1 , wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is favored before amplification of the respective target and disfavored after amplification of the respective target.

3. The method of claim 1 , wherein each energy-transfer probe is configured such that energy transfer from the donor fluorophore to the acceptor fluorophore is disfavored before amplification of the respective target and favored after amplification of the respective target.

4. The method of claim 1 , wherein the step of measuring fluorescence from the energy-transfer probes includes measuring the fluorescence multiple times during the step of amplifying to yield time-dependent fluorescence data.

5. The method of claim 4, wherein the time-dependent fluorescence data from each energy-transfer probe includes an increase in fluorescence from the respective donor fluorophore and a decrease in fluorescence from the respective acceptor fluorophore when the corresponding target is present.

6. The method of claim 4, wherein the time-dependent fluorescence data from each energy-transfer probe includes a decrease in fluorescence from the respective donor fluorophore and an increase in fluorescence from the respective acceptor fluorophore when the corresponding target is present.

7. The method of claim 1, wherein the step of measuring fluorescence includes generating an emission spectrum for each donor fluorophore and each acceptor fluorophore in the mixture, and wherein the step of determining a quantity includes deconvolving the spectra to assess an extent of cleavage for each probe.

8. The method of claim 1, wherein the step of measuring fluorescence includes generating a time-dependent fluorescence intensity curve for each energytransfer probe in the mixture to assess an extent of cleavage for each probe from a baseline representing the concentration of each energy-transfer probe before amplification.

9. The method of claim 1, wherein at least one of the energy-transfer probes further includes a quencher, in addition to a donor and acceptor, wherein the quencher reduces fluorescence from at least one of the donor and acceptor when the probe is intact (i.e., before amplification).

10. The method of claim 9, the energy-transfer probes having an oligonucleotide backbone, wherein the donor, acceptor, and quencher are attached to the backbone with the acceptor disposed between the donor and quencher.11 . The method of claim 9, the energy-transfer probes having an oligonucleotide backbone, wherein the donor, acceptor, and quencher are attached to the backbone with the donor disposed between the acceptor and quencher.

12. The method of claim 9, the energy-transfer probes having an oligonucleotide backbone, the oligonucleotide backbone having two ends, where the donor and acceptor are disposed at or near one end of the backbone and the quencher is disposed at or near the other end.

13. The method of claim 9, the energy-transfer probes having an oligonucleotide backbone, the oligonucleotide backbone having two ends and a middle, where the donor and acceptor are disposed at or near one end of the backbone and the quencher is disposed at or near the middle.

14. The method of claim 1 , wherein a bias correction is applied during signal analysis to account for crosstalk between channels.

15. The method of claim 1 , the mixture including an additional nucleic acid target different from each target in the plurality of distinct nucleic acid targets, further comprising: providing a quencher probe specific to the additional target, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the additional target; amplifying the additional target in the mixture; measuring fluorescence from the quencher probe; and determining from the measured fluorescence a quantity representative of a level of the additional target.

16. The method of claim 15 wherein the steps of amplifying the plurality of distinct targets and amplifying the additional target are performed simultaneously.

17. The method of claim 16, wherein the steps of measuring fluorescence from the energy-transfer probe for each distinct target and measuring fluorescence from the quencher probe are performed simultaneously.

18. The method of any of claims 15, wherein the quenchable fluorophore is selected from the group of fluorophores used to construct the energy-transfer probes.

19. The method of claim 18, wherein the step of determining from the measured fluorescence a quantity representative of a level of the additional target includes subtracting fluorescence data of the energy-transfer probes from measured fluorescence of each total individual fluorophores that cleaved from quencher probes and / or energy-transfer probes (e.g., using Equation 7).

20. The method of claim 15, the mixture including further additional nucleic acid targets different from each target in the plurality of distinct targets, further comprising performing the steps of providing, amplifying, measuring, and determining using a number of quencher probes less than or equal to the number of distinct fluorophores used to construct the energy-transfer probes.

21. The method of claim 1, wherein the number of energy-transfer probes exceeds the number of distinct fluorophores used in their construction.

22. The method of claim 1 , wherein the distinct targets are from distinct infectious agents.

23. The method of claim 1 , wherein the amplification reagents include one or more primers specific to each distinct or additional target, nucleotide triphosphates, and at least one polymerase.

24. The method of claim 1 , wherein the changes in fluorescence from the respective donor fluorophores and respective acceptor fluorophores are changes in an intensity of fluorescence emitted by each fluorophore.

25. The method of claim 1 , wherein the level of each target is selected from the group consisting of a presence or absence of each target, a concentration of each target, and a copy number of each target.

26. The method of claim 1 , wherein the energy-transfer probe includes at least two respective donor fluorophores and a respective acceptor fluorophore, each donor fluorophore capable of energy transfer to the respective acceptor fluorophore.

27. The method of claim 1 , wherein the energy-transfer probe includes at least two respective acceptor fluorophores, each acceptor fluorophore capable of receiving fluorescence excitation energy from the respective donor fluorophore.

28. The method of claim 1 , wherein at least one of the energy-transfer probes and / or quencher probes, when present, is a linear hydrolysis probe.

29. The method of claim 1 , wherein at least one of the energy-transfer probes and / or quencher probes, when present, is a molecular beacons probe.

30. The method of claim 1 , wherein at least one of the energy-transfer probes and / or quencher probes, when present, has an oligonucleotide backbone, and wherein at least one of the probes includes a ribose base doped into its backbone.31 . The method of claim 1 , wherein a 3’-end of at least one probe is blocked.

32. The method of claim 1 , wherein the nucleic acid targets are selected from the group consisting of deoxyribonucleic acid (DNA) targets and ribonucleic acid (RNA) targets.

33. The method of claim 1 , further comprising dividing the mixture for digital PCR analysis prior to the step of amplifying the plurality of targets in the mixture, the targets are present in some but not all the partitions, the targets are amplified in each partition, and the fluorescence from each probe for each target is measured in each partition.

34. A method of performing a multiplexed digital assay, the method comprising: forming a mixture including(i) a sample containing one or more of a plurality of distinct nucleic acid targets,(ii) reagents sufficient for amplification of the targets, and(iii) an energy-transfer probe specific to each target in the first set of targets, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target; dividing the mixture into a plurality of partitions such that a given target is present in some but not all the partitions; amplifying the targets present in each partition; measuring fluorescence from the energy-transfer probe for each target; and determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.

35. The method of paragraph 34, wherein the steps of measuring and determining include assessing whether each partition is positive or negative for each target and assigning a concentration of each target based on a ratio of the number of partitions positive for each target to the total number of partitions.

36. A method of performing a multiplexed assay, the method comprising: providing a mixture including(i) a sample containing one or more of a plurality of distinct nucleic acid targets including a first set of targets and a second set of targets,(ii) reagents sufficient for amplification of the targets,(iii) an energy-transfer probe specific to each target in the first set of targets, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target, and(iv) a quencher probe specific to each target in the second set of targets, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the additional target; amplifying the plurality of targets in the mixture; measuring fluorescence from the energy-transfer probe for each target in the first set of targets and from the quencher probe for each target in the second set of targets; and determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.

37. The method of claim 36, wherein at least one of the energy-transfer probes further includes a quencher.

38. A kit for performing a multiplexed assay for a plurality of nucleic acid targets, comprising: an energy-transfer probe specific to each target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target.

39. The kit of claim 38, further comprising at least one quencher probe specific to an additional target, wherein the quencher probe includes a quenchable fluorophore and a quencher capable of quenching fluorescence from the quenchable fluorophore, wherein the quencher probe is configured such that fluorescence from the quenchable fluorophore is lower before amplification of the additional target and higher after amplification of the additional target, so that changes in fluorescence from the quencher probe can be used to assess the degree of amplification of the additional target.

40. The kit of claim 38, further comprising amplification reagents.41 . The kit of claim 40, wherein the amplification reagents include one or more primers specific to each distinct (or additional target, if applicable), nucleotide triphosphates, and at least one polymerase.

42. A system for performing a multiplexed assay for a plurality of nucleic acid targets, comprising: a quantitative nucleic acid amplification instrument configured to amplify nucleic acid, excite fluorescence from fluorophores, and detect fluorescence emitted by the fluorophores before, during, and / or after amplification; reagents sufficient for amplification of the targets, andan energy-transfer probe specific to each target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, the donor and acceptor fluorophores on a given energy-transfer probe having distinguishable excitation spectra and distinguishable emission spectra, wherein each energy-transfer probe is configured such that an extent of energy transfer from the donor fluorophore to the acceptor fluorophore is altered by amplification of the respective target, so that changes in fluorescence from the donor and acceptor fluorophores can be used to assess the degree of amplification of the respective target.

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