WO2025207981A1 - Tethered probes for multiplexed nucleic acid amplification assays - Google Patents

Abstract

Systems, including methods and compositions, for performing multiplexed assays. The methods may include (A) providing a mixture including (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) a tethered probe specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the tethered 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 tethered probe may include respective first and second fluorophores, with distinguishable excitation and/or emission spectra, and a respective quencher. The probes are configured such that an intensity of fluorescence from one or both fluorophores is altered during amplification of the respective target, so that changes in fluorescence can be used to assess the degree of amplification of the respective target.

Description

TETHERED PROBES FOR MULTIPLEXED NUCLEIC ACID AMPLIFICATION ASSAYS

Cross-References to Priority Applications

[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. The number 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 molecularly tethered probes, each labeled with two or more fluorophores, to enable much higher plex assays than possible using conventional probes labled with a single fluorophore. The probe design may allow modulation of the signal intensity of fluorophores on a given tethered 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) a tethered probe specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the tethered 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 tethered probes may each include respective first and second fluorophores, with distinguishable excitation and/or emission spectra, and a respective quencher. The probes are configured such that an intensity of fluorescence from the first and/or second fluorophore is altered during amplification of the respective target, so that changes in fluorescence from the first and/or second fluorophores can be used to assess the degree of amplification of the respective target. For example, the probes may be configured such that fluorescence from the first and/or second fluorophore is disfavored before amplification of the respective target, due to the presence of the quencher, and favored after amplification of the respective target. In some embodiments, the respective first and second fluorophores and respective quencher may be bound to a common oligonucleotide before amplification and separated from one another when the common oligonucleotide is cleaved during amplification. In some embodiments, the systems may further include one or more quencher probes, each comprising a fluorophore and quencher, where changes in fluorescence of the quencher probes report on respective targets. Exemplary tethered 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 is a schematic showing a standard 5’ nuclease quencher probe, an associated single-target amplification assay, and expected signals obtained with the assay.

[0007] FIG. 2 is a schematic showing a novel tethered probe, an associated single-target amplification assay, and expected signals obtained with the assay.

[0008] FIG. 3 is a standard qPCR plot showing fluorescence as a function of amplification cycle for a standard quencher probe in the presence of target. [0009] FIG. 4 is a dual qPCR plot showing fluorescence intensity (top) and fluorescence intensity increment (bottom) as a function of amplification cycle for each fluorophore on a tethered probe in the presence of target.

[0010] FIG. 5 is a set of plots showing schematic results of a tethered probe assay for three simultaneously present targets and how the signals can be processed to identify the targets under various conditions.

[0011] FIG. 6 is a pair of qPCR amplification plots showing assay results generated at 10, 100, and 1 ,000 target copies per reaction using (A) a standard quencher probe having a single fluorophore (FAM), and (B) a tethered probe having two fluorophores (FAM and ROX). Expected results are obtained with a single FAM signal and signature (synchronized) dual FAM/ROX signals derived from the standard quencher and tethered probes, respectively.

[0012] FIG. 7 is a pair of qPCR amplification plots showing assay results generated using (A) a FAM-Q-ROX tethered probe and (B) a ROX-Q-FAM tethered probe, under otherwise identical assay conditions. The fluorescence intensity for a given fluorophore is different when the fluorophore is positioned at the 3' end of the probe versus the 5' end of the probe. The two probes have the same nucleic acid sequence (SEQ ID NO:1 ), with different fluorophores (FAM and ROX, and ROX and FAM, respectively) at the 5’ and 3’ ends, and with the same quencher (BHQ2) bound in both cases to the eighth base from the 5’ end.

[0013] FIG. 8 is a pair of qPCR amplification plots showing results from a single fluorophore at two different probe concentrations. Doubling the probe concentration (at least approximately) doubles the maximum (plateau-level) fluorescence intensity. [0014] FIG. 9 is a pair of qPCR amplification plots showing assay results generated using (A) a first ROX-Q-FAM tethered probe and (B) a second ROX-Q-FAM tethered probe in which guanine (G) bases have been added immediately adjacent both fluorophores, under otherwise identical assay conditions. The guanine base significantly reduces fluorescence from the 3' FAM fluorophore while only minimally affecting fluorescence from the 5' ROX fluorophore. The two probes have nearly the same nucleic acid sequences (SEQ ID NO:2 and SEQ ID NO:3), differing only in the additional guanine bases in the G+CK+G probe relative to the CK probe. In both cases, the same fluorophores, ROX and FAM, are bound to the 5’ and 3’ ends, respectively, and the same quencher, BHQ1 , is bound to the ninth or tenth base from the respective 5’ ends. The template sequence (SEQ ID NO: 4) is the same as the probe CK sequence but with AGAGTAA added to the 5’ end and GAACTGC added to the 3’ end.

[0015] FIG. 10 is a pair of qPCR amplification plots showing assay results generated using a ROX-Q-FAM probe in which (A) Q is Black Hole Quencher 1 (BHQ1 ) or (B) Q is Black Hole Quencher 2 (BHQ2), under otherwise identical assay conditions. The maximum (plateau-level) fluorescence intensities differ slightly between the two assays, as expected with different quenchers, but both assays show the same sensitivities to target concentrations. The two probes have the same nucleic acid sequence (SEQ ID NO:1 ), with the same fluorophores, ROX and FAM, bound to the 5’ and 3’ ends, respectively, but different quenchers, BHQ1 and BHQ2, bound to the eighth base from the respective 5’ ends.

[0016] FIG. 11 is a pair of qPCR amplification plots showing assay results generated using two different tethered probes, each having a different pair of fluorophores and a different quencher, and each directed to a different target, where the quencher on both probes is positioned very close to the 5' fluorophore. In both cases, fluorescence from the 5' fluorophore is greatly reduced or eliminated, whereas fluorescence from the 3' fluorophore is seemingly normal. The functional fluorophore accurately reports on assay progress and target concentration in both cases.

[0017] FIG. 12 is a set of three qPCR amplification plots showing assay results generated from three different tethered probes (H31-P1 , H31-P3, and H31-P4), each having a different pair of fluorophores but the same quencher, and each directed to the same target, where the quencher on each probe is positioned (just barely) in the 3' half of the probe. In all three cases, fluorescence from the 3' fluorophore (FAM or HEX) is greatly reduced or eliminated, whereas fluorescence from the 5' fluorophore is normal. Results for three additional tethered probes sharing the same quencher position (H31-P5, H31-P6, and H31-P7) and sequence length are similar. Thus, the functional fluorophore accurately reports on assay progress and target concentration in all six cases.

[0018] FIG. 13 is a set of three qPCR amplification plots showing assay results generated using three different probes, each having a different pair of fluorophores and the same or a different quencher, and each directed to a different target, where the quencher on each probe is positioned in the 5' half of the probe (but near the middle). In all three cases, fluorescence from both fluorophores is normal. Both fluorophores accurately report on assay progress and target concentration in all three cases. The drawing is split over two pages due to its size, with two plots and other materials on the first page and a single plot and other materials on the second page). [0019] FIG. 14 is a set of cross-titration curves for two human papillomavirus (HPV) serotypes, HPV56 (FAM and ROX labeled tethered probe) and HPV58 (Cy5 labeled quencher probe), in the presence of 10 to 10⁷ copies per qPCR reaction.

[0020] FIG. 15 is a schematic of a singleplex proximity ligation assay (PLA) for protein detection and quantification.

[0021] FIG. 16 is a pair of excitation and emission spectra for the large Stokes- shift fluorophores ATTO 430LS (top) and ATTO 490LS (bottom). Each fluorophore is excited in a respective excitation channel (ATTO 425 and FAM excitation channels, respectively), but the emission is detected in a different emission channel (SUN and Cy5 emission channels, respectively), red shifted relative to the usual emission channels for the excitation channels.

[0022] FIG. 17 is a schematic showing the use of universal forward primer in multiplex tethered probe assays.

[0023] FIG. 18 is a schematic diagram showing a novel duplex two-fluorophore tethered probe and an associated duplex tethered probe assay.

[0024] FIG. 19 is a set of singleplex qPCR amplification plots comparising the performance of tethered and duplex tethered probes in assays for HPV31 and HPV66. For each HPV in these plots, the respective tethered probe and one of the two duplex tethered probes have the same sequence (SEQ ID NO:5 and SEQ ID NO:7 for HPV31 and HPV66, respectively), while the others of the two duplex tethered probes have complementary sequences (SEQ ID NO:6 and SEQ ID NO:8 for HPV31 and HPV66, respectively). The probes may, more generally, have any suitable sequences. The locations of fluorophores and quenchers on these probes is shown in the figure.

[0025] FIG. 20 is a schematic diagram showing an exemplary multiplexed multi- omics assay directed to colon cancer detection.

[0026] FIG. 21 is a schematic diagram showing an exemplary multiplexed gene expression assay direct to breast cancer stratification. Detailed Description

[0027] The present disclosure provides systems, including methods and compositions, for performing multiplexed assays. These systems use molecularly tethered probes, each labeled with two or more fluorphores, to enable much higher plex assays than possible using conventional probes labled with a single fluorophore. The probe design may allow modulation of the signal intensity of fluorophores on a given tethered 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) a tethered probe and two or more primers specific to each target, (B) amplifying the plurality of targets in the mixture, (C) measuring fluorescence from the tethered 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 tethered probes may each include respective first and second fluorophores, with distinguishable excitation and/or emission spectra, and a respective quencher. The probes are configured such that an intensity of fluorescence from the first and/or second fluorophore is altered during amplification of the respective target, so that changes in fluorescence from the first and/or second fluorophores can be used to assess the degree of amplification of the respective target. For example, the probes may be configured such that fluorescence from the first and/or second fluorophore is disfavored before amplification of the respective target, due to the presence of the quencher, and favored after amplification of the respective target. In such embodiments, the tethered probes each may further comprise a respective oligonucleotide having a sequence complementary to that of a portion of the respective target, where the respective first and second fluorophores and quencher are bound to the oligonucleotide closely enough together that the quencher reduces fluorescence from the fluorophores before amplification, and where the oligonucleotide is cleaved during amplification, separating one or both of the first and second fluorophores from the quencher and causing the fluorescence from the separated fluorophore(s) to increase. In some embodiments, the systems may further include one or more quencher probes each comprising a fluorophore and quencher, where changes in fluorescence of the quencher probes report on respective targets. 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. Exemplary tethered probe applications include infectious disease assays (including HPV assays), proximity ligation assays, multi-omics assays (including early cancer detection), syndromic testing (including respiratory syndromic testing), transplant monitoring, and gene expression assays (including cancer stratification), among others.

[0028] The present disclosure describes both principles and embodiments of tethered probe systems. Examples are presented showing how tethered probes may be used to construct 16-, 22-, 29-, and 37-plex assays from 7, 8, 9, and 10 fluorophores, respectively, among others, with one fluorophore acting as a control. Examples also are presented showing how mixed sets of tethered probes and quencher probes may be used to construct 22-, 29-, 37-, and 46-plex assays from 7, 8, 9, and 10 fluorophores, respectively, among others, again with one fluorophore acting as a control. Assay results may be decoded using fluorophore spectra (colors) and details of the respective qPCR amplification curves. Various functional tethered probes and sets of tethered probes and combinations of tethered probes and quencher probes are presented. These may include oligonucleotides (complementary to respective targets) having a first fluorophore at or nearer the 3' end, a second fluorophore at or nearer the 5' end, and a quencher disposed between the first and second fluorophores. Mechanisms for “tuning” the behavior of the tethered probes are also presented. For example, brightness may be adjusted by altering fluorophore position: fluorophores are generally brighter when placed toward the 3' end of the probe than when placed toward the 5' end. Brightness may be further adjusted by adding G or A bases at the 3' end of the probe, among other modifications, and/or by adjusting probe concentrations, among others. In addition, the absolute and relative brightnesses of the fluorophores may be adjusted by altering the type, position, and/or number of quenchers. One or more of these tuning mechanisms may be used to create unique “signatures” for each tethered probe (such as the ratio of fluorescence from each fluorophore on the probe). [0029] Preliminary results from an exemplary 24-plex human papillomavirus (HPV) assay show a 6-log dynamic range of detection, a 10 copies/reaction limit of detection (LoD), exclusivity, unreduced functionality in the presence of potential interferents, repeatability, positive and negative predictive values (PPVs and NPVs) of 100%, in the presence of 0, 1 , 2, or 3 targets in a sample and serotype calling accuracies of 100% with contrived low-complexity clinical samples based on analysis of fluorescence colors and intensities.

[0030] Tethered-probe assays may have a variety of benefits and advantages. These are presented below, sometimes generally, sometimes in the context of specific applications, and sometimes in comparison with existing assay systems. Many of these advantages arise from the molecular linkage or tethering between the two fluorophores on tethered probes. This linkage creates special features for both encoding and decoding. For example, the linkage facilitates encoding by allowing creation of unique fluorophore combinations that can greatly exceed the number of fluorophores. The linkage also facilitates decoding because the signals from the two tethered fluorophores on a given tethered probe are coupled, meaning, for example, that the ratio of their intensities, and the ratio of the rate at which their intensities increase, may be fixed. Moreover, these ratios may be “tuned” by quencher placement, nearest neighbors (such as adding a guanine (G) base adjacent the 3’ fluorophore).

[0031] 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

[0032] 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.

[0033] 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.

[0034] “Amplification” is a process whereby multiple copies are made of an amplicon matching, complementary to, and/or otherwise corresponding to a target sequence. 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.

[0035] “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.

[0036] “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. [0037] “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 polymer or 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%.

[0038] “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. [0039] 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 or partitions, 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.

[0040] “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.

[0041] “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.

[0042] “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.

[0043] “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.”

[0044] 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.).

[0045] An “HPV” is a human papillomavirus (HPV), the most common sexually transmitted infection (STI) in the United States.

[0046] “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.”

[0047] 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 composed of any suitable number of nucleotides, such as at least about 5, 10, 100, or 1000, among others.

[0048] 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.

[0049] 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.

[0050] 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, tethered probes, energy-transfer probes, and control probes. Oligonucleotides used in probes may be labeled with at least one fluorophore (e.g., quencher probes and control probes), at least two fluorophores (e.g, tethered probes and energy-transfer probes), and/or at least one quencher (e.g., quencher probes, tethered 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 be conjugated 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.

[0051] 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 (see Example 15), among others.

[0052] 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 (e.g, quencher probes, tethered probes, control probes, and some energy-transfer probes) or second fluorophore (e.g., tethered probes and energy-transfer probes). (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,” “tethered probe,” “energy-transfer probe,” and “control probe” import the additional meanings and characteristics described elsewhere in the present disclosure. Moreover, “energy-transfer probe” imports the additional meanings, characteristics, and structures disclosed in the various priority patent applications recited in above under “Cross-References to Priority Applications” and incorporated herein by reference. [0053] 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.

[0054] 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, preamplification, 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.

[0055] 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.

[0056] “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).

[0057] 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.

[0058] 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

[0059] This section provides a brief overview of standard quencher-probe assays, as a counterpoint, and the novel tethered probe assays and mixed tethered probe and quencher probe assays, and mixed tethered probe and quencher probe and energy-transfer probe assays provided by the present disclosure.

[0060] 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. 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.

[0061] FIG. 2 shows a novel tethered 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 tethered probe, which in this embodiment 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. The oligonucleotide may include any suitable number of bases, including, among others, between about 5 and 200 bases, between about 7 and 100 bases, between about 9 and 75 bases, or between about 15 and 45 bases, among others. The tethered probe further comprises first and second fluorophores and a quencher. In short, instead of a single fluorophore and quencher, the tethered probe includes two distinct fluorophores and a quencher. The tethered probe is constructed so that the quencher will measurably, and typically almost completely, reduce fluorescence from the first and second fluorophores. Thus, prior to amplification, excitation of either fluorophore will lead to little or no fluorescence from either fluorophore. However, after amplification and the associated cleavage of the energy-transfer probe, excitation of each fluorophore will lead to fluorescence from that fluorophore. Thus, with each round of amplification and associated degradation of tethered probe, the amount of unquenched first and second fluorophore rises, which is reflected in an increase in fluorescence from each (when each is excited). In essence, the tethered probes effectively create new fluorophores from existing fluorophores, because the synchronized dual fluorescence emission after cleavage differs from that of one fluoropohore alone. The schematic fluorescence signal shown in FIG. 2 corresponds to situations in which both fluorophores are cleaved from one another and from the quencher. However, for reasons explored in the Examples (below), the signal more generally may include four possible scenarios: (1 ) no signal from fluorophore 1 or fluorophore 2; (2) signal from fluorophore 1 but not fluorophore 2; (3) signal from fluorophore 2 but not fluorophore 1 ; and (4) signals from both fluorophore 1 and fluorophore 2.

[0062] A significant advantage of tethered 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 tethered probes that can be created from the same set of fluorophores may be much larger due to combinatorics. See Example 2. Specifically, sets of 3, 4, 5, 6, 7, 8, 9, and 10 distinct fluorophores can only be used to create 3, 4, 5, 6, 7, 8, 9, and 10 distinct quencher probes, respectively. However, sets of 3, 4, 5, 6, 7, 8, 9, and 10 distinct fluorophores can be used to create up to 3, 6, 10, 15, 21 , 28, 36, and 45 distinct tethered probes, respectively. Thus, while tethered 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 seven targets). 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 tethered probes. In some embodiments, tethered probes, or combinations of tethered probes and quencher probes, may be used together with a dedicated control probe. See Examples 3 and 4. In such embodiments, one fluorophore may only be used once (for the control probe). Nevertheless, sets of 6, 7, 8, 9, and 10 distinct fluorophores may be used to create sets of up to 11 , 16, 22, 29, and 37 probes (for a mixture of tethered probes and the control probe) and up to 16, 22, 29, 37, and 46 probes (for a mixture of tethered and quencher probes and the control probe).

[0063] Significantly, despite producing more complex signals, tethered probe assays, mixed tethered probe and quencher probe assays, and mixed tethered probe and quencher probe and/or energy-transfer probe assays can be performed using existing amplification instruments. More specifically, the assays can be performed using the same 3, 4, 5, 6, 7, 8, or more excitation and emission channels used with quencher-probe assays, even though they may include many more probes, each with a unique optical signature determined by the particular combination of 3’ and 5’ fluorophore. 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 the targets. Methods for deconvolving signals and determining target concentrations are described in the next section.

III. Signal Analysis

[0064] This section describes exemplary algorithms for deconvolving the fluorescence signal in amplification assays employing tethered probes or mixed sets of tethered probes and quencher probes (with the latter more complex than the former).

111. A. Probe Deconvolution

[0065] Tethered probes covalently link two (or more) fluorophores on a single probe. The utility of tethered probes in qPCR and other assays is based, in part, on their re-use of the same fluorophores on multiple probes, creating more probes than the number of fluorophores, and allowing analysis of more targets than the number of channels in a qPCR instrument. See Examples 2-4. The combination of tethered probes and quencher probes allows even greater multiplexing. See Examples 3 and 4. This section compares results obtained from standard quencher assays with results obtained from multiplexed tethered-probe assays and shows how the latter can be “deconvolved” to provide information on the absence, presence, and/or concentration of targets in highly multiplexed systems.

[0066] FIG. 3 shows schematic results for a standard single-plex assay, such as the one shown in FIG. 1 , for a specific target using a standard quencher probe, AQ. Here, A is a fluorophore, and Q is a quencher. Each round of amplification cleaves a progressively larger amount of probe, freeing the fluorophore from the quencher. Eventually, enough free (unquenched) fluorophore is created to generate a detectable fluorescence signal. The fluorescence continues to increase sigmoidally. The initial increase in fluorescence is geometric; however, as amplification continues and probe and/or other reagents are consumed, the rate decreases and then asymptotes to zero. The rate at which fluorescence initially becomes detectable is a measure of the concentration of the target: more target, more rapid initial detection; less target, less rapid initial detection. The rate is quantified using cycle number. Specifically, the number of amplification cycles when fluorescence is first detected above a baseline threshold value is called “Ct.” Fluorescence signals plateau at a value ARn.

[0067] The extrapolation of the single-plex analysis to multiplexed quencherprobe assays is straightforward. Multiplex assays use separate quencher probes, AQ, BQ, CQ, etc., for each target, each probe having a unique fluorophore A, B, C, etc. Assays are run by adding all desired probes to the sample, together with the required reagents, and then running the amplification cycles. Fluorophores are excited as amplification proceeds, typically serially, and the resulting fluorescence, if any, is detected. Values of Ct may be used to determine target concentrations, including relative concentrations, if appropriate. The values of Ct for the various fluorophores will be uncorrelated with one another, just with the concentrations of the respective targets. Thus, signals can be deconvolved, and target identity (or identiies) assigned, based solely on color.

[0068] FIG. 4 shows schematic results for a singleplex assay, such as the one shown in FIG. 2, for a specific target using a tethered probe, AQB, instead of a quencher probe. Here, A and B are fluorophores, and Q is a quencher. Each round of amplification again cleaves a progressively larger amount of probe, now freeing two fluorophores instead of just one from the quencher. Eventually, enough of each free (unquenched) fluorophore is created to generate detectable fluorescence signals from each. The two free fluorophores are generated, or released, in an at least approximately constant (e.g., one-to-one (1 :1 )) ratio. Therefore, because the fluorophore ratio is coupled, and thus the number of fluorphore pairs released is coupled, the signals from each fluorophore will behave at least approximately the same.

[0069] The upper panel in FIG. 4 shows traditional qPCR amplification curves plotting fluorescence intensity versus cycle number for each fluorophore on the tethered probe. The cycle number at which the fluorescence signal for each fluorophore first becomes significant relative to background is referred to as Ct. The fluorescence increase from each cycle is referred to as dR. The cumulative sum of each dR is equal to ARn at the plateau. The signals for each fluorophore will have the same or similar Ct values (sometimes differing slightly because one fluorophore generates a stronger signal than the other).

[0070] The lower panel in FIG. 4 shows dR versus cycle number (approximately the 1^st derivative of the fluorescence intensity). The dR values for the two fluorophores on a given tethered probe will be synchronized, moving up and down together and reaching their maxima (signal increase maximum, SIM) at the same cycle (signal inflection point, SIP). Moreover, the cumulative signals will reach their respective plateau values, ARn, after the same number of cycles. The ratio of the two fluorophore’s ARn values is approximately constant at different target abundance, which is defined by properties of the fluorophores, the sequence of the probe, and the relative locations of the fluorophores and quencher.

[0071] The various relationships shown in FIG. 4 for fluorescence from the two fluorophores on a given tethered probe shown may be summarized as follows:

• dR_z reachs a maximum at the same cycle number (the inflection point of the curves, SIP);

• dR, ratio (= dR_{i> FluorophoreA}/dR_{i> Fluorophore B}) is constant at different cycles around SIP, where signal dominates over background; and

• LdR (= ARn) ratio is constant, where the value of the constant is defined by properties of the florophores, the sequence of the probe, and the relative locations of the fluorophores and quencher.

[0072] The extrapolation of the singleplex analysis to multiplex tethered-probe assays, and to multiplex assays involving mixes of tethered probes and quencher probes, is complicated. This complexity, and how it is successfully addressed, can be illustrated with an example. Consider three tethered probes, D1QD2, D3QD2, and D1QD3, formed using fluorophores Di, D2, and D3 and quencher Q. (Q can be the same quencher or different quenchers from probe to probe.) These probes are directed to Targets 1 , 2, and 3, respectively. Table I shows high-level results expected for this system. If only Target 1 is present, fluorescence will be detected only from fluorophores A and B. If only Target 2 is present, fluorescence will be detected only from fluorophores A and C. If only Target 3 is present, fuorescence will be detected only from fluorophores B and C. However, if any two targets are present, or if all three targets are present, then fluoresence will be detected from all three fluorophores A, B, and C.

Table I. Schematic signals produced by a three-fluorophore, three-tethered probe assay for three targets.

Thus, when only a single target is present, signals can be deconvolved, and target identity assigned, based solely on colors. However, when more than one target is present, signals can be deconvolved and target identities assigned by looking at both colors and other aspects (e.g., intensities) of the fluorescence.

[0073] FIG. 5 shows schematic signals for the three-fluorophore, three-target system of Table I when all three targets are present under three sets of conditions (i.e., for the most complicated scenarios). Here, each fluorophore is present on two different probes, and so each fluorophore reports on the presence and concentration of two different targets. No fluorophore reports solely on the presence and concentration of a single target. Therefore, data are measured and plotted for each fluorophore (not for each probe). The top row shows traditional qPCR amplification plots for each fluorophore, the middle row shows dR versus cycle number (the approximate 1^st derivative of the amplification curves) for each fluorophore, and the bottom row shows dR ratios versus cycle number for each pair of fluorophores. The data further correspond to three different scenarios: (A) the three targets have different abundances and different Cts (left column), (B) two of the three targets have similar abundances and similar Cts and thus show overlapping data (middle column), and (C) all three targets have similar abundances and similar Cts and thus they all show overlapping data (right column). When all three targets have different abundances, the amplification curves (top row) collectively show three distinct regions of growth, with two fluorophores increasing together for each target, and each two-fluorophore pair identifies the corresponding target’s existence in the sample. (The amplification curve for each fluorophore shows two growth phases, one for each target on which it reports.) The identification is further confirmed by the signature ARn, dR trend, and SIM of each fluorophore (middle row), and the trend of dR ratio (bottom row), in the probe of the identified target. However, when two or three targets are at similar abundance, it is no longer as easy to identify the targets by the growth phase(s) of the Ct amplification curves. In these cases, targets may be identified by the regression of ARn, SIM, and dR ratio.

[0074] The same mechanisms may be used to determine the presence and concentrations of targets in more complicated assays (e.g., with more probes, more targets, etc.). Decoding is primarily based on (A) the presence of detectable signals from tethered or single fluorophores (when quencher probes are present); (B) the signal inflection point (SIP) and signal increment maximum (SIM) of the respective qPCR curves; (C) the ARn and ARn ratios between the different fluorophores; (D) the trend of dR ratio and its plateau value; and (E) the Ct and ACt between the different fluorophores. Table II provides definitions and additional information regarding these terms and quantities. The use of tethered probes for encoding and decoding facilitates the deconvolution. Specifically, the intramolecularly linked pairs of fluorophores in a tethered probe result in constant ratios of fluorophores’ ARn and SIM. Decovolution is largely based on combinatorial fluorophores and ratios of their signals. Moreover, the coupling synchronizes various changes for a given probe: the fluorescence intensities of tethered fluorophores become detectable against background at the same cycle (Ct), reach their respective maxima (ARn) at the same cycle, reach their maximum rates of change per cycle (SIM) at the same cycle, and have a unique dR ratio trend (plateaus to a constant at cycles near SIP). These couplings and synchronicities make it possible to tease out or otherwise deduce the signals from individual probes from the aggregate signals.

Table II. Definitions of terms and quantities used in the deconvolution of signals in tethered probe assays.

[0075] The relationship among the following quantities can be expressed mathematically:

Here, the first term represents the observed signal in each channel, the second term represents every target signal in each channel, and the third term represents the targets in the sample (where 6_k = 0,1). Data from tethered probe assays may be fitted using this expression to determine the presence or absence of targets. The deconvolution results may be further validated using SIM and dR ratio.

[0076] Table III provides additional highlights of the algorithms used to deconvolve signals from tethered probe assays:

Table III. Highlights of algorithms used to deconvolve signals from tethered probe assays. The details depend on whether a given fluorophore is found on only one probe or on probes for multiple targets.

IV. Examples

[0077] The following examples describe further aspects of the present disclosure, including tethered probes, mixtures of tethered probes and other (e.g., quencher and/or energy-transfer) probes, and associated assays, among others. 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.

Example 1 - Comparison of Tethered Probes and Quencher Probes

[0078] This example compares results obtained using dual-fluorophore tethered probes and standard single-fluorophore quencher probes; see FIG. 6. The fluorophores are FAM and ROX; the quencher is Black Hole Quencher 1 (BHQ1 ). The tethered probe structure is 5'-FAM-BHQ1-ROX-3'. The quencher probe structure is 5'- FAM-BHQ1. Amplification assays are run at three target concentrations: 10, 100, and 1 ,000 copies per reaction. The results of both sets of assays agree, at all three target concentrations, confirming that the tethered probe accurately reports on both the progress of amplification and the presence and concentration of target.

Example 2 - Probe Combinatorics (Basic)

[0079] This example describes basic aspects of probe combinatorics, specifically, the numbers and types of two-fluorophore, one-quencher tethered probes that can be constructed using a given number of distinct fluorophores; see Table IV. The case in which one such fluorophore is set aside for use as a control is described in Example 3. Fluorophores and quenchers are denoted using alphabetical designators: (1 ) fluorophores are denoted using A, B, C, D, E, F, G, H, I, and J, and (2) quenchers (of whatever type) are denoted using Q. (The quenchers used may or may not vary from probe to probe, depending on what quenchers are most effective with given fluorophores.)

Table IV. Exemplary tethered probes that can be constructed using up to ten unique fluorophores. Here, fluorophore order and quencher identity within a given probe are ignored. More generally, any suitable number of fluorophores can be used, consistent with the measurement capabilities of the associated instrument in the associated assay. [0080] The maximum possible number, N, of unique tethered probes that can be constructed from n distinct fluorophores is given by the following equation: n(n — 1) N ⁼ 2

This number can be doubled (i.e., N = n(n - 1)) if fluorophore order is taken into account in probe construction (e.g., if a probe having a 5' A and a 3' B (AQB) and a probe having a 3' A and a 5' B (BQA), among others, behave differently). However, because such “mirror image” probes are not usually used in the same assay, they do not (typically) affect the maximum degree of multiplexing for a given number of fluorophores. Table IV shows probe configurations that can be constructed using up to 10 unique fluorophores. Larger probe sets may be constructed using the same enumerative (or other applicable) approach. Any suitable subset of the maximum number of possible probes may be used in any given assay.

Example 3 - Probe Combinatorics (Enhanced w/ Internal Control)

[0081] This example describes enhanced aspects of probe combinatorics, specifically, the numbers and types of two-fluorophore, one-quencher tethered probes that can be constructed using a given number of distinct fluorophores, where one fluorophore is set aside for use as a control (e.g., an “internal control”); see Tables V and VI. Fluorophores and quenchers are again denoted using alphabetical designators: (1 ) fluorophores used in tethered probes are denoted using A, B, C, D, E, F, G, and H, (2) the fluorophore used as a control is denoted using Z, and (3) quenchers (of whatever type) are denoted using Q.

Table V. Exemplary tethered probes that can be constructed using up to ten unique fluorophores, where one fluorophore is set aside as a control. Here, like in Table IV, fluorophore order and quencher identity within a given probe are ignored. More generally, any suitable number of fluorophores can be used, consistent with the measurement capabilities of the associated instrument in the associated assay.

[0082] The maximum possible number, N , of unique probes that can be constructed from n distinct fluorophores, where one fluorophore is set aside as a control, is given by the following equation:

Here, the first term is the number of tethered probes, and the second term is the number of control probes (in this case, unity). This number can, in principle, be (approximately) doubled (i.e., N = (n - l)(n - 2) + 1) if fluorophore order is taken into account in probe construction (i.e., if AQB BQA). However, as discussed in Example 2, this does not (typically) affect the maximum degree of multiplexing. Table V shows probe configurations that can be constructed using up to 10 unique fluorophores. Larger probe sets may be constructed using the same enumerative (or other applicable) approach. Any suitable subset of the maximum number of possible probes may be used in any given assay.

[0083] Table VI shows three exemplary sets of tethered probes constructed from 7 (left), 8 (middle), and nine (right) fluorophores, respectively. Here, the fluorophores are specifically identified: FAM, ROX, Cy5, VIC, TMR, ATTO 425, ATTO 490LS (8 and 9 fluorophore sets only), ATTO 430LS (9 fluorophore set only), and Cy5.5 (the control fluorophore). More generally, the sets may be constructed using any suitable fluorophores capable of being distinguished under the conditions of the assay. The quenchers may be the same or different, from probe to probe.

A: 7 -dye Set: 16-plex max

C: 9-dye Set: 29-plex max

Table VI. Exemplary (A) 16, (B) 22, and (C) 29-member sets of tethered probes constructed from 7, 8, and 9 fluorophores, respectively, where one fluorophore (Cy5) is set aside as an internal control (IC).

Example 4 - Mixed Probe Sets

[0084] This example describes exemplary mixed probe sets comprising combinations of tethered probes, quencher probes, and/or energy-transfer probes; see Tables VII and VIII. Fluorophores and quenchers are denoted using the same alphabetical designators (A, B, C, D, E, F G, H, Z, and Q) used in Example 3 (without, like in Examples 2 and 3, considering the possibility that different quenchers may be used in different probes). The maximum possible number, N, of unique probes in a mixed probe set formed from combinations of tethered probes and quencher probes that can be constructed from n distinct fluorophores, where one fluorophore is set aside as a control, is given by the following equation: (n — l)(n — 2)

N = - - - - + (n - 1) + 1

2

Here, the first term is the number of tethered probes, the second term is the number of quencher probes, and the third term is the number of control probes (in this case, unity). This number can, in principle, be significantly increased (i.e., N = (n - l)(n - 2) + (n - 1) + 1 ) if fluorophore order is taken into account in probe construction (i.e., if AQB BQA. However, as discussed in Example 2, this does not (typically) affect the maximum degree of multiplexing. Table VII shows probe configurations that can be constructed using up to 10 unique fluorophores. Larger probe sets may be constructed using the same enumerative (or other applicable) approach. Any suitable subsets of the tethered and quencher probes may be used in a given assay. More specifically, some of the tethered probes and/or some of the quencher probes may be omitted, with the total number of probes corresponding to the desired level of multiplexing. Moreover, the number of dedicated control fluorophores could be increased (here and in Example 3). In some cases, larger numbers of fluorophores may be used (e.g., 10, 11 , 12, or more) in probe construction, depending on the capabilities of the instrument(s) used in the assay.

Table VII. Exemplary mixed set of tethered probes and quencher probes that can be constructed using up to ten unique fluorophores, where one fluorophore is set aside as a control. Here, like in Examples 2 and 3, fluorophore order and quencher identity within a given probe are ignored. More generally, any suitable number of fluorophores can be used, consistent with the measurement capabilities of the associated instrument in the associated assay.

[0085] Table VIII shows three exemplary mixed sets of tethered and quencher probes constructed from 7 (left), 8 (middle), and nine (right) fluorophores, respectively. Here, the fluorophores are specifically identified: FAM, ROX, Cy5, VIC, TMR, ATTO 425, ATTO 490LS (8 and 9 fluorophore sets only), ATTO 430LS (9 fluorophore set only), and Cy5.5 (the control fluorophore). More generally, the sets may be constructed using any suitable fluorophores capable of being distinguished under the conditions of the assay. The quenchers may be the same or different, from probe to probe. A: 7 -dye Set: 22-plex max

B: 8-dye Set: 29-plex max

C: 9-dye Set: 37-plex max

Table VIII. Exemplary (A) 22, (B) 29, and (C) 37-member sets of mixed tethered and quencher probes constructed from 7, 8, and 9 fluorophores, respectively, where one fluorophore (Cy5) is set aside as an internal control (IC).

[0086] Tethered probes also may be combined, depending on assay goals, with energy-transfer probes (or both quencher and energy-transfer probes), as well as any desired control probes. Energy-transfer probes include donor and acceptor fluorophores having distinct excitation and/or emission spectra. The donor and acceptor fluorophores can be excited separately using excitation light of the appropriate wavelength(s). However, if the donor and acceptor are sufficiently close, such as when bound together on an energy-transfer probe, an excited donor fluorophore, instead of fluorescing, may instead transfer its excitation energy non- radiatively to the nearby acceptor fluorophore, which then fluoresces instead at a different, typically lower, wavelength. Energy-transfer probes are designed such energy transfer occurs before amplification and not after amplification. Exemplary energy-transfer probes are described in U.S. Provisional Patent Application Serial No. 63/570,776, filed March 27, 2024, and titled “Energy-Transfer Probes for Multiplexed Nucleic Acid Amplification Assays.” Tethered probes may be combined with energytransfer (and other) probes following the logic underlying Tables VII and VIII.

Example 5 - Effects of Fluorophore Position

[0087] This example explores effects of fluorophore position on fluorescence intensity in tethered-probe assays; see FIG. 7. The fluorophores are FAM and ROX; the quencher is Black Hole Quencher 2 (BHQ2). Two tethered probes are used, one with FAM in the 5' position and ROX in the 3' position, the other with ROX in the 5' position and FAM in the 3' position. Both probes target HPV56 and are identical except for fluorophore position. Amplification assays are run, under identical conditions, at three target concentrations. The amplification curves from each fluorophore in each assay are different; however, the underlying results are the same. In particular, for a given target concentration, the signal thresholds and signal plateaus are reached after the same numbers of cycles for both probes and both fluorophores. The signal for a given fluorophore may be higher or lower when the fluorophore is in the 3' versus than 5' position on tethered probes, depending (at least in part) on the location of the quencher. In some cases, the signal amplitudes are higher for a given fluorophore when the fluorophore is in the 3' rather than 5' position. In other cases, the signal amplitudes are higher for the fluorophore when the fluorophore is in the 5' rather than 3' position. Thus, fluorophore position (together with quencher position) may be used to adjust the level of the ARn plateau and thus to create distinguishable probes from the same two fluorophores.

Example 6 - Effects of Probe Concentration

[0088] This example explores effects of probe concentration on fluorescence intensity in tethered-probe assays; see FIG. 8. A single tethered probe is used targeting HPV39. Amplification assays are run under identical conditions, except for the concentration of tethered probe. The concentrations of tethered probe are 40 nM (left panel) and 80 nM (right panel). The cycle thresholds (Ct) at both probe concentrations are similar. However, the ARn plateau values scale with probe concentration: doubling the probe concentration, from 40 nM to 80 nM, doubles the plateau values, from about 75,000 to about 150,000 for ROX and from about 30,000 to about 60,000 for Cy5 in the units of the assay.

Example 7 - Effects of Fluorophore Environment

[0089] This example explores effects of fluorophore environment, specifically, nearest-neighbor bases, on fluorescence signals of a given assay; see FIG. 9. Two tethered probes are used, each targeting HPV56. Both probes include an oligonucleotide, a 5' ROX, a 3' FAM, and a Black Hole Quencher 1 (BHQ1 ). The oligonucleotides are identical except that one of the probes further includes a guanine (G) base at each end of the oligonucleotide next to the respective fluorophores. Amplification assays are run, under otherwise identical conditions, using each probe. Fluorescence from the 3' FAM is significantly reduced when a nearest-neighbor G is present. In contrast, fluorescence from the 5' ROX is relatively unaffected by a nearest-neighbor G. Thus, fluorescence from a 3' fluorophore, such as FAM, may be tuned by adding one or more 3' guanine bases adjacent the fluorophore. The same or similar effects may be achieved using other suitable moieties, such as an adenine (A) base (or bases)

Example 8 - Effects of Quencher Identity

[0090] This example explores effects of quencher identity (or type) on assay performance for two otherwise identical tethered probes; see FIG. 10. Both probes target HPV56 and have identical oligonucleotides, fluorophores (5' ROX and 3' FAM), and fluorophore and quencher positions (both quenchers are at the 8^th base from 5’ end fluorophore (ROX)). However, one probe (left panel) uses Black Hole Quencher 1 (BHQ1 ), whereas the other probe (right panel) uses Black Hole Quencher 2 (BHQ2). These quenchers differ in their absorption spectra. Specifically, BHQ1 preferentially absorbs between about 480 and 580 nm, whereas BHQ2 preferentially absorbs between about 559 and 670 nm. Assays are run, under otherwise identical conditions, at three target concentrations. While the signal strengths obtained in both sets of assays vary slightly, reflecting differences in the quenchers, the abilities of both probes and both sets of assays to detect target reproducibly and to distinguish target concentrations are the same. Thus, either quencher can be paired with the fluorophores used in this example.

Example 9 - Effects of Quencher Position

[0091] This example explores effects of quencher position on fluorescence intensity in tethered-probe assays; see FIGS. 11-13. In three separate sets of assays, quenchers are positioned near the 5' end of tethered probes, the 3' half of tethered probes, and the middle of the 5' half of tethered probes. The final position generates the most robust probe performance.

[0092] FIG. 11 shows exemplary amplification curves for two different tethered probes having quenchers positioned close to the 5' end of each probe. Specifically, in this example, the respective fluorophores are positioned at the 5' (FAM and ROX) and 3' (TAMRA and ATTO 425) ends of the probes, and the respective quencher is positioned either three (BHQ1 ) or four (BHQ2) bases from the 5' ends of the probes. One probe targets HPV31 (FAM and TAMRA); the other probe targets HPV11 (ROX and ATTO 425). Assays are run, under otherwise identical conditions, at two target concentrations: 10 and 300 copies per reaction. In all cases, little or no signal is collected from the fluorophores at the 5' ends of the probes (i.e., near the quenchers), whereas significant signal is collected from the fluorophores at the 3' ends of the probes (i.e., far from the quenchers). A possible explanation is that during amplification the polymerase fails to cleave the probe between the 5' fluorophore and quencher when the two are very close together. In this case, fluorescence from the 5' fluorophore will remain quenched, even after amplification. However, the DNA polymerase does cleave the probe between the quencher and the 3' fluorophore, rendering the 3' fluorophore fluorescent. Thus, in these assays, the tethered probes are behaving like single-fluorophore quencher probes, based on fluorescence from the respective 3' fluorophores.

[0093] FIG. 12 shows six different tethered probes having identical lengths and having quenchers positioned in the 3' half of each probe, together with exemplary amplification plots for three of the probes. (The amplification plots for the other three probes are similar.) Specifically, in this example, the respective fluorophores are again positioned at the 5' (Cy5, FAM, and ROX) and 3' (FAM, HEX, TAMRA, and Cy5) ends of the probes, and the quencher (in all cases BHQ1 ) is positioned in the 3' half of the probes (in all cases, 17 bases from the 5' end and 11 bases from the 3' end). All probes target HPV31. Assays are run for six different probes, under otherwise identical conditions, at two target concentrations: 20 and 100 copies per reaction. In all cases, little or no signal is collected from fluorophores at the 3' ends of the probes, whereas significant signal is collected from fluorophores at the 5' ends of the probes. A possible explanation is that during amplification the probes release from the target before cleavage occurs between the quencher and 3'-fluorophore, leaving the two moieties bound to the same remnant of the probe and the 3' fluorophore therefore quenched. Thus, in these assays, the tethered probes are behaving like single-fluorophore quencher probes, based on fluorescence from the respective 5' fluorophores.

[0094] FIG. 13, which is spread over two pages, shows exemplary amplification plots for three different tethered probes having quenchers positioned at or near the middle of the 5' halves of the probes, at least 8 bases away from the 5' end. A total of thirty-seven different probes have been examined with similar results, under otherwise identical conditions, directed to a variety of targets and using a variety of fluorophores and quenchers. Target concentrations include 10, 100, 1 ,000, and 10,000 copies per reaction. For each probe, and for each target concentration, significant signal is collected from both fluorophores on the probe. Moreover, the amplification curves display the expected behaviors based on Ct and ACt values in the presence of target molecules at 10 to 10,000 copies per reaction. In addition, amplification plots show very similar ARn values with an input from 10 to 10,000 copies per reaction. ARn ratios of fluorophore pairs are 1 :1 with HPV35 (FAM/Cy5), 1 :2 with HPV45 (FAM/ROX), and 2.5:1 with HPV56 (FAM/ROX), respectively. Because the ARn ratios of a given fluorophore pair may be tuned by altering fluorophore position, quencher position, and/or the presence or absence of one or more A or G bases at the 3’ position, among others, it may be possible to decode HPV45 and HPV56 based on their distinct ARn ratios even though they are labeled with same fluorophore pair. Example 10 - Exemplary Applications 1 : 12-Plex HPV Assays

[0095] This example describes the design of an exemplary 12-plex assay for the detection and identification of human papillomavirus (HPV) variants using a mixture of tethered, quencher, and control probes; see Table IX. The assay employs six fluorophores. One fluorophore (Cy5.5) is used once as an internal control (IC). The remaining five fluorophores (FAM, ROX, Cy5, HEX, and TAMRA) are used multiple times to construct five quencher probes (out of a possible five that can be constructed from five fluorophores) and six tethered probes (out of a possible ten that can be constructed from five fluorophores). This assay may be run using an instrument having at least six excitation and emission channels.

Table IX. Fluorophore selection for a 12-plex HPV assay.

Example 11 - Exemplary Applications 2: 24-Plex HPV Assays

[0096] This example describes the design and testing of an exemplary 24-plex assay for the detection and identification of human papillomavirus (HPV) serotypes or variants using a mixture of both tethered and quencher probes; see Tables X to XII and FIG. 14. In particular, the example describes assay design and a series of tests exploring assay exclusivity, sensitivity to interference, limit of detection, repeatability, dynamic range, and accuracy.

[0097] Assay design. Tables X-XII summarize the design of the assay, including both materials and methods. Table X shows the HPV serotypes that can be identified using the assay and the 24 associated probes, which include a mixture of tethered probes and quencher probes. The assay employs seven fluorophores. One fluorophore (Cy5.5) is used once as an internal control (IC). The remaining six fluorophores (ATTO 425, FAM, ROX, Cy5, VIC, and TAMRA (TMR)) are used multiple times to construct six quencher probes (out of a possible six that can be constructed from six fluorophores) and seventeen tethered probes (out of what would appear to be a possible fifteen that can be constructed from six fluorophores). However, in the case of the tethered probes, three tethered probes share the same two fluorophores (ATTO 425 and TMR) tuned such that each probe produces distinguishable signals (e.g., unique ARn ratios). This assay may be run using an instrument having at least seven excitation and emission channels.

Table XI. qPCR reaction mixture. Table XII. qPCR cycling protocol. (Fluorescence signals are collected at stage 3 at 62°C step.)

The samples tested include (1 ) synthetic plasmids for the evaluation of analytical performance, (2) contrived clinical samples for the evaluation of clinical performance, and (3) six homologous microorganisms (Ureaplasma urealyticum, Neisseria gonnorrhoeae (Gonococcus), Candida albicans, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis) and human genomic DNA (gDNA) for exclusivity and interference studies. The reaction mixtures include (1) 24 pairs of target-specific primers at 25-120 nM each, (2) a universal forward primer at 400 nM (see Example 15), and (3) 24 unique probes at 25-150 nM each, including 3 probes having the same fluorophore pair: ATTO 425 and TMR, targeting HPV42, HPV43, and HPV81 , respectively. Table XI shows components of the reaction mixture, which include a probe/primer mix, a buffer mix, an enzyme mix, and a template. Table XII shows the cycling amplification protocol, including temperatures and cycle durations. Runs are performed using an Applied Biosystems QuantStudio 5 Real-Time PCR System (52 minutes with 5+40 cycles).

[0098] Exclusivity. Table XIII shows results of exclusivity testing. The goal is to show that the assay does not deliver positive results when target is absent. Here, the assay is run on samples containing six homologous microorganisms and human genomic DNA (gDNA) as a positive control, but no HPV, at concentrations of 10⁵ to 10⁷ copies/mL. The six microorganisms employed are recommended for exclusivity testing by the Chinese National Medical Products Administration (NMPA). The results suggest that the HPV assay is specific because under these conditions no signal was observed.

Table XIII. Exclusivity study of 24-plex HPV assay.

[0099] Sensitivity to interference. Table XIV shows results of interference testing. The goal is to show that the assay correctly reports the presence of target despite the further presence of potentially interferents. Here, the HPV assay is run in the presence of (1) HPV51 at 20 copies/reaction (the target), using a Cy5/ROX tethered probe, and (2) the six microorganism strains used in the exclusivity study, again at 10⁵ to 10⁷ copies/mL (the potential interferents). The results show no detectable interference (i.e., the assay correctly detects HPV51 despite the presence of the six microorganism strains).

Table XIV. Interference study of 24-plex HPV assay. [0100] Limit of Detection. Table XV shows results of limit-of-detection testing. The goal is to determine assay sensitivity, i.e., the lowest concentrations of target at which target is still reproducibly detectable. Here, the HPV assay is run for 23 HPV targets at 10 target copies/reaction. The results show that all targets are consistently (-100%) detectable at this concentration, suggesting that the limit of detection is less than or equal to 10 targets copies/reaction, whether with quencher probes or tethered probes.

[0101] Repeatability. Table XVI shows results of repeatability (also called reproducibility) testing. The goal is to show that assay results are reproducible (i.e., that repeated measurements generate the same result with high confidence). Here, the HPV assay is run ten times for three targets — HPV33, HPV56, and HPV51 — at 50 target copies/reaction. A quencher probe is used for one target, HPV33, and tethered probes are used for two targets, HPV56 and HPV51. The table shows Ct for each target for each run. The tight range of standard deviations (Stdev: 0.18 to 0.41 ) and coefficients of variation (CV: 0.6% to 1.4%) for a given fuorphore in a given assay suggest that these HPV assays are reproducible. The observed differences in Ct between different fluorophores and different HPV serotypes are expected.

Table XVI. Repeatability study of 24-plex HPV assay.

[0102] Dynamic range. Table XVIIAB and FIG. 14 show results of dynamic range testing. The goal is to show a range of target concentrations over which the assay correctly reports the presence, type, and relative concentration of targets. Here, cross-titration assay results are shown for two targets present in each sample, one increasing in concentration from sample to sample, the other decreasing in concentration from sample to sample. The targets are HPV56 and HPV58. Table XVI IA shows results for HPV56, based on two fluorophores: FAM and ROX. Table XVI IB shows results for HPV58, based on one fluorophore: Cy5. Cross-titrations between HPV56 and HPV58 are plotted from 10 to 10⁷ copies per reaction; see FIG. 14. At a given target concentration, the number of cycles required to reveal target is the same for both targets. Moreover, the number of cycles required for detection is linear on a semi-log plot. The results, taken together, show that the assays have at least a 6-log (10⁶-fold) dynamic range, meaning that they work at very low, intermediate, and very high target concentrations in the presence of two different targets.

Table XVIIA. Cross-titration results for HPV56 in 24-plex HPV assay.

[0103] Accuracy. Table XVIII shows results of accuracy testing. The goal is to show that the assay correctly reports the presence and type of target. Here, assays are run with and without target, and results are summarized using both positive and negative predictive values and serotyping accuracy. Positive predictive value (PPV) (or positive percent agreement (PPA)) means that the assay gives a positive result when the target is present. In clinical terms, it means that a patient with a positive test result truly has the pathogen or disease. Negative predictive value (NPV) (or negative percent agreement (NPA)) means that the assay gives a negative result when the target is absent. In clinical terms, it means that a patient with a negative test result is truly free of the pathogen or disease. Serotyping accuracy means that the positive test correctly identifies the underlying serotype. In clinical terms, a positive result for a target only occurs when that exact target is present and not when the assay confuses a related target for the exact target (e.g., a positive result for HPV56 means that HPV56 is present and not, for example, HPV58). The results suggest that the assays are accurate: PPV was 100% (30/30 positive results) in the presence of 1 , 2, or 3 HPV targets or hits; NPV was 100% (17/17 negative results); and serotyping accuracy was 100% (30/30 correct results) in contrived low-complexity clinical samples.

Table XVIII. Initial clinical evaluation of 24-plex HPV assay using contrived samples at 100% PPV, 100% NPV, and 100% serotyping accuracy.

Example 12 - Exemplary Applications 3: Proximity Ligation Assays

[0104] This example describes exemplary multiplexed proximity ligation assays for sensitive protein identification and/or quantification; see FIG. 15. The proximity ligation assay (PLA) uses a pair of target-specific antibodies per each target protein. The antibodies are linked with DNA (or other nucleic acid) strands, which serve as reporter sequences to form proximity probe pairs. These probe pairs may simultaneously and pairwise bind to their respective target analytes, bringing them into proximity and enabling an enzymatic ligation reaction of the DNA (or other nucleic acid) strands. The assays may be run in solution and/or any other suitable medium. The ligated probes form a new PCR amplicon composed of both reporter sequences of the proximity probes. This reporter molecule reflects the identity of the protein through sequence encoding in multiplex, and its amount corresponds to the protein analyte concentration. By attaching multiple pairs of reporter sequences to multiple target-specific antibodies, multiple proteins can be analyzed in a single multiplex PLA reaction combined with a multiplex PCR/qPCR reaction. The multiplex PLA assays disclosed here may include a set of 2-50 (commonly 15-30) pairs of reporter oligonucleotides each conjugated with target-specific antibody pairs. Ligated nucleic acid products of the multiplex PLA reaction can be amplified and decoded using the 2-50 plex tethered probe (or mixed (e.g., tethered, quencher, and/or energy-transfer) probe) qPCR assay, as described elsewhere in the present disclosure. The multiplex tethered and mixed probe PLAs more generally may involve any suitable specific binding partner(s) (e.g., antibodies, Fab fragments, etc.), including mixtures thereof, directed to any suitable target(s) (e.g., proteins, protein digests, macromolecular assemblies, vesicles, etc.). PLA probes allow multiplex-PCR assays to be used to identify and/or quantify protein (or other antibody-recognizable) targets. This ability, in turn, allows multiplex PCR assays to be used to detect mixtures of proteins and nucleic acids, permitting consolidation of assays that would otherwise need to be run separately, reducing complexity and associated costs.

Example 13 - Exemplary Applications 4

[0105] The tethered probes and mixed tethered and quencher probes provided by the present disclosure may be used more generally for any suitable purpose or application in any suitable assay, including but not limited to purposes and assays currently investigated using quencher probes.

[0106] 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 papillomavirus (HPV) (as described, for example, in Examples 10 and 11 ), 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, among others), 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. [0107] 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.

[0108] 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 tethered probes or mixed tethered and quencher probes, fluorescence signals for digital PCR could be measured at the beginning of amplification to determine a starting amount of tethered 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 non-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 14 - Large Stokes Shift Fluorophores

[0109] This example describes how fluorophores having large Stokes shifts can be used to increase the effective number of excitation and emission channels in a qPCR instrument without hardware changes; see FIG. 16 and Table XIX. This increased number of channels, in turn, allows the use of an increased number of probes, including tethered probes, and thus increases the potential “plex” of the assay. Fluorescence, as noted elsewhere in this disclosure, involves the absorption of one or more “excitation” photons by a fluorophore and the fluorophore’s subsequent emission of a lower-energy fluorescence emission photon. The wavelength shift between the shorter-wavelength, higher-energy excitation photon and the longer-wavelength, lower-energy emission photon is known as the “Stokes shift.” Fluorescence instruments, such as qPCR instruments, are set up to excite fluorophores in a first wavelength range (excitation channel) and detect the subsequent fluorescence is a second nonoverlapping wavelength range (emission channel). The wavelength difference between the two channels will at least approximately equal the Stokes shift. Thus, for a given fluorophore, such as FAM, an instrument may have a FAM excitation channel and a FAM emission channel. This combination of channels can be used for FAM or, more generally, for any fluorophores that can be excited and detected using the same channels (i.e., that have excitation and emission spectra similar to FAM). Thus, an instrument having 6, 7, 8, 9, or 10 channels, among others, can be used with 6, 7, 8, 9, or 10 standard fluorophores each absorbing and emitting in a respective one of the channels.

[0110] The number of usable fluorophores can be increased using nonstandard fluorophores, such as large Stokes shift fluorophores, that absorb and emit in nonstandard channel pairings. For example, the large Stokes-shift fluorophore ATTO 490LS can be excited in the standard FAM excitation channel but emits in the standard Cy5 emission channel. Thus, ATTO 490LS and FAM can be used in the same assay because their respective fluorescence emissions will be detected in different, distinguishable channels. ATTO 490LS and Cy 5 can also be used in the same assay because they will be excited in different, distinguishable channels. FIG. 16 shows excitation and emission spectra for ATTO 490LS and ATTO 430LS, another large Stokes shift fluorophore. Table XIX(A) shows how ATTO 490LS can be used to augment seven standard fluorophores to allow detection of eight fluorophores in a seven channel system. In this case, the eight total fluorophores could be used to create 29 total probes, including tethered, quencher, and control probes, measured using just seven channels. Table XIX(B) shows how ATTO 490LS and ATTO 430LS can be used to augment a different but overlapping set of standard fluorophores to allow detection of nine fluorophores in a seven-channel system. In this case, the nine total fluorophores could be used to create 37 total probes, including tethered, quencher, and control probes, again measured using just seven channels. The key, in both cases, is that each fluorophore, standard and nonstandard, is excited and detected using a unique pair of channels. These same principles can be applied to other fluorophores, and fluorophore combinations, and other detection systems having fewer, the same, or even more channels. In each case, the preset channel options for excitation and emission (e.g., FAM/FAM, SUN/SUN, etc.) are augmented by additional options created by exciting in one of the standard excitation channels and detecting in a channel red-shifted by one or more channels from the standard emission channel for the respective excitation channel.

TABLE XIX(A). Exemplary eightTABLE XIX(B). Exemplary nine-color color detection scheme for sevendetection scheme for seven-channel channel qPCR instrument (including qPCR instrument (including two large one large Stokes shift (LS) Stokes shift (LS) fluorophores using fluorophore using unmatched different combinations of unmatched excitation and emission channels). excitation and emission channels).

Example 15 - Universal Forward Primers

[0111] This example describes the use of universal forward primers in multiplexed tethered probe and mixed probe assays; see FIG. 17. The universal forward primer comprises at least a sequence identical, or nearly identical, to a forward primer tail sequence incorporated into the target-specific primers. In assays incorporating a universal forward primer, a target-specific primer binds to the target in the initial or early rounds of amplificaition. The forward primer tail sequence is replicated together with the target sequence, forming an amplicon having the sequence FPS-TS (wherein FPS is a sequence complementary to the forward primer tail sequence, and TS is the target sequence). In subsequent rounds of amplification, the universal forward primer binds to FPS, if present (which will be true only if the target was present), while the target-specific tethered or mixed probe binds to TS as usual. The universal forward primer may be used with linear tethered (or mixed) probes and/or duplexed tethered (or mixed) probes. The use of universal forward primer has several advantages in a multiplex assay:

• Universal primer will enable the reduction of target-specific primer concentrations (e.g., from about 500-1 ,000 nM down to about 25-50 nM, among others), lowering overall primer concentration and reducing primer cost per test;

• Lower overall primer concentration, in turn, will reduce nonspecific primer/primer or primer/probe interactions in general, improving multiplex assay performance; and

• Universal primer will likely yield more even amplifications of all targets in a multiplex assay.

In some embodiments, more than one universal forward primer and/or one or more universal reverse primers may be used.

Example 16 - Duplexed Tethered Probes

[0112] This example describes exemplary double-stranded or “duplexed” tethered probes; see FIGS. 18 and 19. These duplexed probes comprise two complementary oligonucleotides that can bind to one another via base pairing to form a double-stranded duplex. At least one of the oligonucleotides is specific to a desired target sequence. One of the two oligonucleotides (a “fluorophore oligonucleotide”) includes respective first and second fluorophores, with distinguishable excitation and/or emission spectra. The other of the two oligonucleotides (a “quencher oligonucleotide”) includes first and second quenchers. The fluorophores may be bound at any suitable locations along the fluorophore oligonucleotide; however, typically they will be far enough from one another to avoid unintended interactions (e.g., energy transfer) and to allow cleavage during amplification. For example, they may be bound at or near opposite ends of the oligonucleotide. Here, “near” may include positions within about 0-10 bases, among others, from the ends of the oligonucleotide. The quenchers similarly may be bound at any suitable locations along the quencher oligonucleotide; however, typically they will be bound close to the respective fluorophores so they can quench fluorescence from the fluorophores when the duplexed tethered probe is in its double-stranded uncleaved configuration. For example, the quenchers may be bound to or next to a base complementary to the base to which the respective fluorophores are bound. [0113] FIG. 19 is a comparison of singleplex qPCR assay results obtained using tethered and duplex tethered probes for HPV31 and HPV66. Both tethered probes (D1-Q-D2) and duplexed tethered probes (D1-D2 and Q2-Q1) successfully generate qPCR amplification curves with similar Ct values. Compared with tethered probes, in this example, duplexed tethered probes give relatively lower fluorescence signals (ARn) at 3' fluorophores but similar signal levels at 5' fluorophores, possibly due to differences in probe quenching efficiencies.

[0114] The duplexed tethered probes may have advantages relative to their single-stranded oligonucleotide F1QF2 counterparts. For example, they may provide more consistent cleavage during amplification because without the intervening quencher the oligonucleotide will be more available to nuclease activity by the polymerase. This, in turn, may lead to higher fluorescence signals and likely increased assay sensitivity. In addition, the duplexed probes may have better quenching efficiency, because the quenchers can be closer to the respective fluorophores they are intended to quench, and because different, more appropriate quenchers can be used for each fluorophore. In other words, the first and second quenchers adjacent the first and second fluorophores can be the same or different. In contrast, in singlequencher tethered probes, the single quencher will typically be the best compromise effective for both fluorophores. This concept is further discussed in Example 18 (Exemplary Quenchers).

[0115] The duplexed tethered probes have the disadvantage of requiring two labeled probes per target, namely, the two-fluorophore tethered probe and the complementary two-quencher probe, in duplex form.

[0116] Duplexed tethered probes may be used together with and/or in place of single-stranded tethered probes in the various assays and examples described in the present disclosure.

Example 17 - Exemplary Fluorophores

[0117] The PCR probes of the present disclosure, including tethered probes, quencher probes, energy-transfer probes (if any), 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 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, Carbopyronin, 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; TEX 615; SUN; and Tye 563, Tye 665, and Tye 705. The use of large Stokes-shift (LS) fluorophores, such as ATTO 430LS and ATTO 490LS, among others, allows eight- and nine-color detection from existing seven-color qPCR systems; see Example 14. More generally, LS fluorophores can be used to create an additional detection channel for each LS- fluorophore supported by the system.

Example 18 - Exemplary Quenchers

[0118] The PCR probes of the present disclosure, including tethered probes, quencher probes, energy-transfer probes (if any), 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), dabacyl 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, BHQ0 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. Thus, the quencher used in tethered probes having just one quencher may be a compromise choice based on the best average quencher performance for the two fluorophores. Relatedly, the use of a single fluorophore may place limits on which two fluorophores may be used on a given tethered probe. For example, performance may be reduced if one fluorophore emits in the blue and the other fluorophore emits in the red. The quenchers used in tethered probes having two quenchers, such as duplexed tethered probes, may be optimized for each fluorophore. For example, BHQ1 could be matched with a fluorophore emitting in the blue, and BHQ2 or BHQ3 could be matched with a fluorophore emitting the red.

Example 19 - Multi-Fluorophore and Multi-Donor Probe Sets

[0119] This example describes exemplary tethered probes, and tethered probe sets, involving three or more fluorophores and/or two or more quenchers. In some cases, tethered probes may include two or more copies of the same fluorophore. For example, multi-fluorophore probes constructed from fluorophores A and B could include AAQB, AQBB, and AAQBB, among others. In this case, doubling up fluorophores may increase fluorescence signals without altering the character of the signals. In other cases, tethered probes may include two or more quenchers. For example, a two-fluorophore two-quencher probe constructed again from fluorophores A and B could include AQQB. In this case, doubling up quenchers may be more effective than a single quencher at reducing probe fluorescence before amplification. In yet other cases, tethered probes may include more than two fluorophores and more than one quencher. For example, a three-(or more)-fluorophore two-quencher probe could include AAQQB, AQQBB, and AAQQBB, among others. In this case, the extra fluorophores and extra quenchers could increase signal after amplification and/or reduce signal before amplification. Additionally, probes may be constructed using three or more different fluorophores. For example, a probe having fluorophores A, B, and C could include AQBQC, among others. In this case, the probe would generate a fundamentally different fluorescence signature that two-fluorophore tethered probes because up to three (or more) fluorescence signals would be created by amplification. Probes having three or more fluorophores and/or two or more quenchers may be used alone, together with other similar probes, and/or together with two-fluorophore singlequencher probes, quencher probes, energy-transfer probes, and/or control probes, among others.

Example 20 - Deconvolution by dR

[0120] This example describes an exemplary method for deconvolving tethered probe data. The method involves analysis of the signal increment, dR, which is the change in fluorescence signal from a given fluorophore between successive amplification cycles (see, e.g., Table II). For a given fluorophore, dR is the sum of signal changes contributed by all targets with a probe containing the fluorophore:

The terms in this equation are defined as follows:

• Y is the dR matrix (i.e., the dR value of each fluorophore at each cycle) for the sample;

• X, is the dR matrix of target /;

• / is the proportional error of target /; and

• E is the error of detection.

The error terms, <t>,- and E, as assumed to have normal distributions. The data may then be analyzed as follows:

1 . Calculate the likelihood P(YI ) of each possible solution of target combination { t/ }, where P(Ylt,) = Z P(YI ) * P(X/lf/)

{ X/ } is the combination of interpolated results from known targets with various inputs;

2. Get the solution { t, } with maximum likelihood; and

3. Reconstitute the dR curve of each target, and use the curve to calculate Ct values.

Example 21 - Exemplary Sample Holders and Instrumentation

[0121] The multiplexed assays described in the present disclosure may be performed using any suitable sample holders and detection modalities and any suitable amplification and detection device(s).

[0122] 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 sample sites. 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 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.

[0123] 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 22 - Exemplary Applications 5: Multi-Omics and Early Cancer Detection

[0124] This example describes exemplary multiplexed multi-omics tests for simultaneously analyzing nucleic acid, methylation (of nucleic acid), and proteins, among others; see FIG. 20 and Table XX. Exemplary targets may arise in and/or reflect the genome, proteome, transcriptome, epigenome, metabolome, and microbiome, among others. Multi-omics assays may focus on a constellation of nucleic acid and protein targets relating to, or allowing diagnosis and/or characterization of, a condition, such as cancer. For example, constellations of DNA mutations, methylation, and protein biomarkers may be used for the early detection and characterization of cancer, such as colorectal (OR) cancer, among others. The multiplexed multi-omics tests disclosed here use a set of multiplexed PCR probes, such as the tethered probes, energy-transfer probes, and/or quencher probes described herein, each probe specific to nucleic acid reflecting a presence and/or activity of a different multi-omic target. Samples, mixed with probes and other reagents, are subjected to PCR, typically in only one or two sample wells or other holders, in a multiplex format. Fluorescence from the probes is induced, collected, and analyzed as described elsewhere in this disclosure to determine the presence and/or abundance of each multi-omic target. DNA mutations may be detected directly, using appropriate allelespecific primers. DNA methylation may be detected using methylation-specific PCR, for example, by pre-treating potentially methylated DNA with appropriate enzymes and/or using two pairs of primers. Protein can be detected indirectly by creating a corresponding nucleic acid signature that can be detected via PCR, for example, by performing a proximity ligation assay directed to the protein and using the proteinspecific ligate as a target (see Example 12).

[0125] FIG. 20 shows an exemplary multiplexed multi-omics test directed to CR cancer detection. Suitable biomarkers for the text are listed in Table XX and include, but are not limited to, (1) methylation markers, such as NDRG4 and BMP3, among others; (2) mutation markers, such as KRAS mutations, among others; and (3) fecal hemoglobin (representative of fecal occult blood), among other possible protein markers. Initially, a stool (or other suitable) sample is collected. The sample is prepared for multiplexed DNA and protein analysis. DNA is extracted from the sample and a methylation-specific digestion(s) is performed. Multiplexed PCR is performed to characterize methylation and mutation targets in the sample. A hemoglobin-specific proximity ligation assay also is performed on the sample. PCR is then performed to test for the presence and amount of ligate as a measure of the presence and amount of hemoglobin in the sample. Fecal hemoglobin is indicative of bleeding and thus disease, such as precancerous or cancerous polyps, in the colon, bowel, and/or rectum. The DNA subsample and hemoglobin-specific subsample may be analyzed separately, using the same instrument, or combined for PCR analysis together.

Table XX. Exemplary markers for a multiplexed assay directed to colon cancer.

Example 23 - Exemplary Applications 6: Syndromic Testing

[0126] This example describes exemplary multiplexed tests for diagnosing infectious diseases by simultaneously testing for multiple pathogens that may cause similar clinical signs and symptoms; see Table XXI. The use of such “syndromic” tests to identify underlying pathogen(s) allows treatment of causes rather than just the symptoms of an infection. For example, knowing whether the infection is viral or bacterial informs whether to treat with an anti-viral medication or an antibiotic. Moreover, it allows public health experts to track diseases of concern. Exemplary applications include diagnosing respiratory infections, blood infections, gastrointestinal infections, neural infections, and sexually transmitted infections, among others. The multiplexed tests disclosed here use a set of multiplexed PCR probes, such as the tethered probes, energy-transfer probes, and/or quencher probes described herein, each probe specific to nucleic acid reflecting a presence and/or activity of a different pathogen or other infectious agent. Samples, mixed with probes and other reagents, are subjected to PCR, typically in a single sample well or other holder, in a multiplex format. Fluorescence from the probes is induced, collected, and analyzed as described elsewhere in this disclosure to determine the presence and/or abundance of each pathogen. The “plex” of these tests is determined by the number of pathogens being investigated. Exemplary plexes may include 15, 16, 17, 18, 19, 20, 21 , and 22, among others. Tests may be run at the point of care (POC) or at a centralized facility.

[0127] An exemplary application of syndromic testing is to diagnose respiratory infections. Table XXI contains a list of exemplary respiratory pathogens suitable for respiratory syndromic testing. A given test may be directed at nucleic acid representing some or all of these pathogens and/or at other (e.g., newly emerging) respiratory pathogens. Samples may be collected using any suitable mechanism, such as a nasal swab, a nasal wash, and/or a nasal aspirate, among others, and processed to release or otherwise render accessible nucleic acid before performing PCR.

testing.

[0128] Analogous tests can be used to diagnose other infections, including but not limited to blood infections (e.g., sepsis (including resistance markers)), gastrointestinal infections (e.g., norovirus, rotavirus, Campylobacter, salmonella, shigella, Clostridium difficile, E. coli, etc.), neural infections (e.g., meningitis, encephalitis, HIV, progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM), Creutzfeldt-Jakob disease, herpes encephalitis, neurocysticercosis, tropical spastic paraparesis, etc.), and sexually transmitted infections (e.g., HPV, herpes, chlamydia, gonorrhea, HIV/AIDS, syphilis, etc.), among others. [0129] Analogous tests also can be used to diagnose veterinary and/or agricultural infections (e.g., to diagnose respiratory and/or other infections of animals, such as dogs, cats, poultry, and livestock, among others, as well as plants).

Example 24 - Exemplary Applications 7: Transplant Monitoring

[0130] This example describes exemplary multiplexed tests for transplant monitoring, namely, assessing the health of transplanted organs and transplant recipients after transplant surgery and/or identifying potential complications, such as organ rejection. Monitoring, in turn, may allow better assessment of graft (transplant) function and better management of the recipient’s immune system. The multiplexed tests described here use a set of multiplexed PCR probes, such as the tethered probes, energy-transfer probes, and/or quencher probes described herein, each probe specific to nucleic acid reflecting an aspect of the health or function of a transplanted organ and/or the associated recipient. Samples may include blood, urine, and the like. Samples, mixed with probes and other reagents, are subjected to PCR, typically in a single sample well or other holder, in a multiplex format. Fluorescence from the probes is induced, collected, and analyzed as described elsewhere in this disclosure.

[0131] Exemplary nucleic acid targets, or markers, may include, among others, (A) donor-derived cell-free DNA (dd-cfDNA), and/or (B) specific genes related to human immune response. dd-cfDNA is DNA that is released into the blood and interstitial fluid from dead and dying, or otherwise compromised, transplant cells. The concentration of dd-cfDNA can rise shortly after a transplant but should then fall and remain steady if the transplanted organ is healthy. Thus, dd-cfDNA is an important marker for organ health. dd-cfDNA can be distinguished from recipient DNA using human SNP ID markers (e.g., from a database of suitable markers, such as markers in the U.S. National Institutes of References and Technologies (NIST) Standard Reference Database SRD 130, among others). The health of transplanted organs can be adversely affected if the organ is “rejected” or otherwise targeted by the recipient’s immune system. Therefore, assaying the expression of genes involved in an organ recipient’s immune response can indicate both the strength and detailed nature of the response. This information, in turn, can be used to manage the targets and strengths of immunosuppressant medications, among other uses. Example 25 - Exemplary Applications 8: Gene Expression Assays and Cancer Stratification

[0132] This example describes exemplary multiplexed tests for assessing gene expression and exemplary uses of such tests in cancer stratification; see FIG. 21 and Table XXI I. Gene expression is the process whereby the information encoded in genes is converted into function. This typically involves production of RNA, including mRNA, via transcription, and proteins from the RNA, via translation. The types of genes that are expressed (i.e., active) and the extent of that expression may be assayed by determining the presence and amount of RNA corresponding to that gene in a sample. Gene expression assays may be used to diagnose diseases, characterize variations in those diseases, identify potential drug targets specific to diseases and their variations, and/or monitor treatment efficacy, among other uses. The multiplexed tests described here use a set of multiplexed PCR probes, such as the tethered probes, energy-transfer probes, and/or quencher probes described herein, each probe specific to RNA expressed by a particular gene of interest. Samples may include any suitable and appropriate source of RNA. Examples include tissue biopsies, including cancer biopsies, from which RNA can be extracted (and DNA separated or digested). Samples, mixed with probes and other reagents, are subjected to PCR, typically in a single sample well or other holder, in a multiplex format. Fluorescence from the probes is induced, collected, and analyzed as described elsewhere in this disclosure. The results of the assay may be reported as a presence (or absence) of a gene product (meaning that the gene is being expressed), variations or mutations in the gene product, and/or a relative or absolute abundance of the gene product (e.g., a concentration).

[0133] Exemplary gene expression assays may be used in cancer stratification (e.g., tissue type (histology), gene mutations (molecular markers), and the presence of pertinent hormone receptors. Stratification is important because many current anticancer agents are targeted at underlying biological mechanisms. Thus, a detailed molecular knowledge may be a prerequisite to formulating an appropriate treatment strategy. Stratification also may be used to predict the risk of a cancer recurrence (i.e., to provide a long-term prognosis). Suitable cancers for this analysis include breast cancer and gastric cancer, among others. [0134] Breast cancer stratification may be performed using RNA extracted from suitable tumor tissue, such as formalin-fixed paraffin-embedded (FFPE) tissue, among others. Multiplexed assays may be performed with any suitable type and number of RNA markers, including RNA for cancer-related genes and reference genes. Table XXIII shows a suitable set, although other sets can be constructed from a subset of these markers and/or combinations of some or all of these markers with other markers.

Breast Cancer-Related Genes

Reference Genes

TABLE XXII. Twenty-one exemplary cancer-related and reference genes for use in breast cancer stratification.

The expression of these genes may be quantified using the multiplexed assays described herein. For example, Ct values are determined. Ct values for cancer genes may be normalized by subtracting an average value, or other representative value, constructed from Ct values for the reference genes. The numbers obtained can be used to characterize the cancer, to inform treatment, and/or to predict the likelihood of a recurrence based on a suitable “recurrence score,” such as the following:

Recurrence Score = + 0.47 x HER2 Group Score

- 0.34 x Estrogen Group Score

+ 1 .04 x Proliferation Group Score

+ 0.10 x Invasion Group Score

+ 0.05 x CD68

- 0.08 x GSTM1

- 0.07 x BAG1

In general, higher values of the recurrence score correlate with higher likelihoods of recurrence and thus motivate more aggressive treatment regimens. For example, in an exemplary interpretation (and implementation), recurrence scores less than 18 correspond to a low risk of recurrence (such that chemotherapy may not be recommended), values between 18 and 30, inclusive, correspond to an intermediate risk of recurrence (such that chemotherapy should be considered), and values greater than or equal to 31 correspond to a high risk of recurrence (such that chemotherapy is likely to be recommended).

Example 26 - Exemplary Applications 9: Exemplary 15-Plex Assay

[0135] This example describes an exemplary 15-plex assay that uses 10 dyes to label probes for 15 targets according to the pattern shown in the Table XXIII. The intensity of each dye, after the dye-containing probe is fully hydrolyzed (i.e., at the ARn plateau value of the dye when the corresponding target is present in the amplification reaction), is set at two different levels with a relative value of 1 and 0.5 when used to label different targets. The amplification of 14-15 targets coexisting at identical concentration (all targets with identical Ct values, which is extremely rare) is simulated with ARn having a standard deviation of 7% due to the concentration difference of the input targets. The amplification data is decoded using the algorithm specificied herein. The decoding accuracy is >99.9%.

Table XXIII. Exemplary setup for 15-plex assay.

Example 27 - Selected Aspects

[0136] 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. Methods

M. 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) a tethered probe specific to each target, wherein each tethered probe includes a first fluoroohore, a second fluoroohore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second 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 the fluorescence from the tethered 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.

+ Oligonucleotide

MO1. The method of paragraph M, each tethered probe further including an oligonucleotide having a 5' end and a 3' end, wherein the respective first fluorophore is bound to the oligonucleotide at or near the 5' end, the respective second fluorophore is bound to the oligonucleotide at or near the 3' end, and the respective quencher is bound to the oligonucleotide between the 5' and 3' ends.

M01a. The method of paragraph MO1 , wherein the quencher is closer to the 5' end than to the 3' end.

MO1b. The method of claim MO1 or M01a, wherein the respective first fluorophore and second fluorophore are bound to the oligonucleotide within about 0- 10 bases from the 5' end and 3' end, respectively.

MO2. The method of paragraph MO1 or MO1a, wherein the oligonucleotide is hydrolyzed during amplification, such that at least one of the first fluorophore and second fluorophore is separated from the respective quencher, increasing fluorescence from each separated fluorophore.

M02a. The method of paragraph MO2, wherein both the first fluorophore and the second fluorophore are separated from the quencher during amplification, increasing fluorescence from both fluorophores.

MO3. The method of any of paragraphs MO1 to M02a, wherein a same type of fluorophore is used on two distinct tethered probes, each distinct tethered probe being specific to a distinct target.

M03a. The method of paragraph MO3, wherein the intensity of fluorescence from the same type of fluorophore is different from one of the two distinct probes than from the other.

MO3b. The method of paragraph MO3 or M03a, wherein the same type of fluorophore is nearer a 5' end of one of the two distinct tethered probes and nearer a 3' end of the other.

MO3b1 . The method of paragraph MO3b, wherein the intensity of fluorescence from the same type of fluorophore is higher after amplification if the fluorophore is at a 3' end of a probe than if it is at a 5' end of the probe.

MO3b2. The method of paragraph MO3b, wherein the intensity of fluorescence from the same type of fluorophore is higher after amplification if the fluorophore is at a 5' end of a probe than if it is at a 3' end of the probe.

M03c. The method of paragraph MO3 or M03a, wherein the same type of fluorophore is nearer a same end of the two distinct tethered probes, and wherein a guanine (G) base or an adenine (A) base is adjacent the same fluorophore on one of the two distinct tethered probes but not the other.

M03c1 . The method of paragraph M03c, wherein the same type of fluorophore is nearer a 3' end of each of the two distinct tethered probes.

MO3d. The method of paragraph MO3, wherein the same type of fluorophore is used on two distinct tethered probes at or near a same 5’ end or a same 3’ end, and the two distinct tethered probes have different fluorophores at or near their 3’ end or their 5’ end, respectively.

MO4. The method of paragraph M, each tethered probe being a linear probe, wherein the respective quencher is disposed between the respective first and second fluorophores. M04a. The method of paragraph MO4, wherein the respective quencher is closer to one of the respective first and second fluorophores than the other.

MO5. The method of paragraph M, wherein in at least one tethered probe, the quencher is a third fluorophore which serves to quench a level of fluorescence from at least one of the first fluorophore and the second fluorophore.

MO6. The method of paragraph M, wherein at least one of the first fluorophore and the second fluorophore associated with at least one tethered probe is a large stokes-shift fluorophore.

MO7. The method of any preceding paragraph, wherein the nucleic acid targets are intermediate products during protein identification and/or quantification, the presence and/or level of the nucleic acid targets representing the presence and/or level of respective protein targets.

M07a. The method of paragraph MO7, wherein the nucleic acid targets are products from a proximity ligation assay (PLA) reaction during protein identification and/or quantification.

MO7b. The method of paragraph MO7 or M07a, the protein identification and/or quantification assay including a protein target-specific antibody or other protein target-specific partners linked with a reporter sequence, wherein at least one of the nucleic acid targets is formed from the reporter sequence or an amplified copy of the reporter sequence.

M07c. The method of paragraph MO7b, the protein identification and/or quantification assay or the proximity ligation assay (PLA) comprising a set of 2-50 pairs of antibodies linked with reporter sequences, wherein the 2-50 pairs of antibodies are specific to 2-50 plex proteins.

+ Duplex Tethered Probes

DTP. The method of paragraph M, the quencher being a first quencher, further comprising a second quencher, wherein the first and second fluorophores are bound to a first oligonucleotide, and wherein the first and second quenchers are bound to a second oligonucleotide, and wherein a sequence of the first oligonucleotide and a sequence of the second oligonucleotide are at least substantially complementary, allowing the oligonucleotides to base pair and form a duplex. DTP1a. The method of paragraph DTP, wherein the first fluorophore and the first quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

DTP1 b. The method of paragraph DTP1a, wherein the second fluorophore and the second quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

DTP2. The method of any of paragraphs DTP to DTP1 b, wherein the first and second quenchers are different.

DTP2a. The method of paragraph DTP2, wherein the first quencher is BHQ-1 and the second quencher is BHQ-2.

+ Probe Combinatorics

MC1 . The method of any preceding paragraph, wherein the number of tethered probes exceeds the number of distinct fluorophores used in their construction.

MC2. The method of any preceding paragraph, wherein the tethered probes include all pairwise combinations of at least four fluorophores, each pairwise combination reporting on a distinct target.

MC3a. The method of any preceding paragraph, the tethered probes being constructed from distinct fluorophores A and B and a quencher Q, among others, wherein the fluorophores and quencher on one of the tethered probes specific for a first distinct target are AQB, in that order, and the fluorophores and quencher on another of the tethered probes specific for a second distinct target, different from the first, are BQA, in that order.

MC3b. The method of any preceding paragraph, the tethered probes being constructed from distinct fluorophores A and B and a quencher Q, among others, wherein the fluorophores and quencher on two of the tethered probes specific for two distinct targets are in the same order, AQB, and wherein a guanine base is adjacent A or B in one of the tethered probes but not the other.

MC4. The method of any preceding paragraph, the tethered probes being constructed from at least four distinct fluorophores A, B, C, and D, wherein the tethered probes include the following combinations of fluorophores and quenchers (wherein the fluorophores on a given probe are in any suitable order): AQABB, AQACC, AQADD, BQBCC, BQBDD, and CQCDD. MC4a. The method of paragraph MC4, wherein at least some of QAB, QAC, QAD, QBC, QBD, and QCD are the same.

MC4b. The method of paragraph MC4, wherein all of QAB, QAC, QAD, QBC, QBD, and QCD are the same.

MC5. The method of any preceding paragraph, the tethered probes being constructed from at least five distinct fluorophores A, B, C, D, and E, wherein the tethered probes are selected from the following combinations of fluorophores (wherein the fluorophores on a given probe are in an suitable order): AB, AC, AD, AE, BC, BD, BE, CD, CE, and DE.

MC6. The method of any preceding paragraph, the tethered probes being constructed from at least six distinct fluorophores A, B, C, D, E, and F, wherein the tethered probes are selected from the following combinations of fluorophores (wherein the fluorophores on a given probe are in an suitable order): AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, and EF.

MC7. The method of any preceding paragraph, the tethered probes being constructed from at least seven distinct fluorophores A, B, C, D, E, F, and G, wherein the tethered probes are selected from the following combinations of fluorophores (wherein the fluorophores on a given probe are in an suitable order): AB, AC, AD, AE, AF, AG, BC, BD, BE, BF, BG, CD, CE, CF, CG, DE, DF, DG, EF, EG, and FG.

MC8. The method of any preceding paragraph, further comprising an additional fluorophore used only once and not present on a tethered probe.

+ Third Fluorophore and/or Second Quencher

MT1. The method of any preceding paragraph, wherein at least one of the tethered probes includes a third fluorophore.

MT1a. The method of paragraph MT1 , wherein the excitation and emission spectra of the third fluorophore are the same as the excitation and emission spectra of either the first fluorophore or the second fluorophores.

MT1 b. The method of paragraph MT1 , wherein the excitation and emission spectra of the third fluorophore are distinct from the excitation and emission spectra of the first and second fluorophores.

MT2. The method of any preceding paragraph, wherein the tethered probe further includes a second quencher. + Quencher Probes

MQ. The method of any preceding paragraph, the mixture including at least one 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 each additional target, wherein each quencher probe includes a different 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 respective additional target and higher after amplification of the respective additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the respective additional target; (B) amplifying each additional target in the mixture; (C) measuring fluorescence from each respective quencher probe; and (D) determining from the measured fluorescence a quantity representative of a level of each additional target.

MQ1a. The method of paragraph MQ, wherein the quenchable fluorophores are selected at least in part from the group of fluorophores used to construct the tethered probes.

MQ1b. The method of paragraph MQ or MQ1a, wherein the quenchers used to construct the quenchable probes are selected at least in part from the group of quenchers used to construct the tethered probes.

MQ2. The method of any of paragraphs MQ to MQ1 b, wherein the steps of amplifying the plurality of distinct targets and amplifying each additional target are performed simultaneously.

MQ3. The method of any of paragraphs MQ to MQ2, wherein the steps of measuring fluorescence from the tethered probes and quencher probes are performed by measuring the fluorescence from each fluorophore in the system, independent of how many probes include the fluorophore.

+ Energy-Transfer Probes

MET. The method of any preceding paragraph, the mixture including at least one additional nucleic acid target different from each target in the plurality of distinct nucleic acid targets, further comprising (A) providing an energy-transfer probe specific to each additional target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, wherein the 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 additional target; (B) amplifying each additional target in the mixture; (C) measuring fluorescence from each respective energy-transfer probe; and (D) determining from the measured fluorescence a quantity representative of a level of each additional target.

MET1. The method of paragraph MET, wherein the donor and acceptor fluorophores are selected from the group of fluorophores used to construct the tethered probes.

MET2a. The method of paragraph MET or MET1 , 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.

MET2b. The method of paragraph MET or MET1 , 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.

MET3. The method of any of paragraphs MET1 to MET2, wherein the steps of measuring fluorescence from the tethered probes and energy transfer probes are performed by measuring the fluorescence from each fluorophore in the system, independent of how many probes include the fluorophore.

+ Measuring

MME. The method of any preceding paragraph, wherein the step of measuring fluorescence from the probes includes measuring the fluorescence multiple times during the step of amplifying to yield time-dependent fluorescence data.

MME1. The method of paragraph MME, wherein measuring the fluorescence multiple times corresponds to measuring the fluorescence after each amplification cycle (in other words, after each round of amplification).

MME2. The method of paragraph MME or MME1 , wherein measuring the fluorescence multiple times corresponds to measuring the fluorescence across different emission wavelengths (e.g., using different detection channels). MME3. The method of any of paragraphs MME to MME2, wherein the timedependent fluorescence data from each tethered probe includes an increase in fluorescence from the respective first and second fluorophores when the corresponding target is present.

MME4. The method of paragraph MME3, wherein the increase in fluorescence from the respective first and second fluorophores is linked.

MME4a. The method of paragraph MME4, wherein a ratio of the rate of change in fluorescence of the first fluorophore and the rate of change in fluorescence of the second fluorophore is at least approximately constant during amplification.

MME5. The method of any preceding paragraph, wherein the target is identified by the colors of the fluorescence from the respective tethered probe when only one target is present in the mixture.

MME6. The method of any preceding paragraph, wherein the targets are identified by both the colors and intensities of the fluorescence from the respective tethered probes when the tethered probes for more than one target share a same fluorophore.

MME6a. The method of paragraph MME6, wherein the targets are identified by a synchronized change in fluorescence intensity at each amplification cycle.

MME6a1 . The method of paragraph MME6a, wherein the synchronized change involves at least one of Ct, a signal inflection point, a dR ratio, and a AR ratio.

+ Digital Assays

DA. 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) a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second 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 the fluorescence from the tethered probe for each target in each partition; and (E) determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.

DA1. The method of paragraph DA, 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.

DA2A.The method of paragraph DA or DA1 , wherein the steps of forming a mixture and dividing the mixture are performed simultaneously.

DA2B.The method of paragraph DA or DA1 , wherein the step of forming a mixture is performed before the step of dividing the mixture.

D3. The method of any of paragraphs D1 to DA2B, further comprising the additional step(s) and/or limitation(s) from any of claims M to MME6a1 .

+ Miscellaneous

MMIO. The method of any preceding paragraph, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.

MMI1. 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.

MMI2. The method of any preceding paragraph, further comprising a fluorophore (e.g., Cy5.5) that is used only once.

MMI2a. The method of paragraph MMI2, wherein the fluorophore that is used only once is used on a quencher probe.

MMI3. The method of any preceding paragraph, wherein the fluorophores are selected from the group consisting of FAM, ROX, Cy5, VIC, TMR, ATTO 425, ATTO 430LS, ATTO 490LS, and Cy5.5.

MMI4. The method of any of paragraphs M to MMI3, 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.

MMI4a. The method of paragraph MMI4, wherein the level of each target is a presence or absence of each target. MMI4b. The method of paragraph MMI4, wherein the level of each target is a concentration of each target.

MMI4c. The method of paragraph MMI4, wherein the level of each target is a copy number of each target.

MMI5. The method of any preceding paragraph, the multiplexed assay being a multiplexed qPCR assay, wherein the step of amplifying the targets includes performing a polymerase chain reaction.

MMI6. The method of any preceding paragraph, the quencher being a third fluorophore, wherein the quencher reduces fluorescence from at least one of the first and second fluorophores, and wherein fluorescence from the quencher, if detected from at all, is distinguishable from fluorophore fluorescence.

MMI7. The method of any preceding paragraph, 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.

+ Indications

IND1. The method of any of paragraphs M to MMI7, wherein the nucleic acid targets are selected from the group consisting of deoxyribonucleic acid (DNA) targets and ribonucleic acid (RNA) targets.

INDIA. The method of paragraph IND1 , wherein the nucleic acid targets are deoxyribonucleic acid (DNA) targets.

IND1 B. The method of paragraph IND1 , wherein the nucleic acid targets are ribonucleic acid (RNA) targets.

IND1 B1. The method of paragraph IND1 B, wherein the multiplexed assay is a gene expression assay.

IND1 B1a. The method of paragraph IND1 B1 , wherein the gene expression assay is used in cancer (e.g., breast cancer) stratification.

IND1C. The method of any of paragraphs M to MMI6, wherein the nucleic acid targets are a mixture of deoxyribonucleic acid (DNA) targets and ribonucleic acid (RNA) targets. IND1 D. The method of any of paragraphs M to MMI6, wherein the nucleic acid targets are donor-derived cell-free DNA (dd-cfDNA) targets (e.g., for transplant monitoring).

IND1 D1. The method of paragraph IND1 D, wherein the donor-derived cell-free DNA (dd-cfDNA) targets include single-nucleotide polymorphism (SNP) markers.

IND1 E. The method of any of paragraphs M to MMI6, wherein the nucleic acid targets include, or are derived from, methylated nucleic acid.

IND1 E1 . The method of paragraph IND1 E, wherein the methylated nucleic acid is methylated deoxyribonucleic acid.

IND1 F. The method of any of paragraphs M to MM16, wherein the nucleic acid targets are selected from a group consisting of at least two of methylation markers, mutation markers, and protein markers.

IND1 F1 . The method of paragraph IND1 F, wherein the nucleic acid targets are selected to assess colon cancer risk.

IND1 G. The method of any of paragraphs M to MM16, wherein the nucleic acid targets are selected to diagnose a disorder from a group of disorders sharing similar symptoms (e.g., syndromic testing).

IND1 G1. The method of paragraph IND1 G, wherein the group of disorders is selected from a further group consisting of respiratory infections, blood infections, gastrointestinal infections, neural infections, and sexually transmitted infections.

IND1 G2. The method of paragraph IND1 G or IND1 G1 , wherein the group of disorders is selected from a further group consisting of human disorders, veterinary disorders, and agricultural disorders.

B. Kits

K. A kit for performing a multiplexed assay for a plurality of nucleic acid targets, comprising a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second fluorophores can be used to assess the degree of amplification of the respective target.

K1 . The kit of paragraph K, further comprising amplification reagents sufficient for amplification of the targets.

K1a1. The kit of paragraph K1 , 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.

K1 b. The kit of paragraph K1 or K1a1 , wherein the amplification reagents include one or more forward primers specific to each distinct (or additional target, if applicable), and one or more universal forward primers specific to each of the forward primers, or one or more universal forward primers with sequence identical (or complementary) to the tail sequence of each of the forward primers.

K2. The kit of any preceding kit paragraph, further comprising at least one quencher probe.

K3. The kit of any preceding kit paragraph, further comprising at least one energy-transfer probe.

K4. The kit of any preceding kit paragraph, wherein the quencher comprises a third fluorophore having fluorescence that is distinguishable from the fluorescence of the first and second fluorophores.

K5. The method of any of paragraphs K to K4, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.

C. Systems

S. 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) a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second fluorophores can be used to assess the degree of amplification of the respective target.

51. The system of paragraph S, wherein the quantitative nucleic acid amplification instrument is a qPCR or dPCR instrument.

52. The system of paragraph S or S1 , wherein the quantitative nucleic acid amplification instrument includes excitation and emission filters to preferentially excite and detect fluorescence from a desired fluorophore from the group of fluorophores used to the probes.

53. The system of any of paragraphs S to S2, wherein the quantitative nucleic acid amplification instrument includes a stage configured to support a multiwell plate or reaction chambers on a chip.

S3a. The system of paragraph S3, further comprising a multiwell plate or chip.

54. The system of any of paragraphs S to S2, wherein the quantitative nucleic acid amplification instrument includes a droplet generator for partitioning the mixture for digital PCR amplification.

55. The system of any of paragraphs S to S4, further comprising one or more quencher and/or energy-transfer probes.

S6. The method of any of paragraphs S to S5, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.

D. Tethered Probes

P1. A tethered probe for multiplexed nucleic acid amplification assay comprising (1 ) a first fluorophore; (2) a second fluorophore, having distinguishable excitation spectra and/or distinguishable emission spectra with the first fluorophore; and (3) a quencher capable to quench a level of fluorescence from the first fluorophore and/or the second fluorophore when probe is intact; wherein the tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first fluorophore and the second fluorophore is altered between when the probe is intact and cleaved.

P2. The tethered probe of paragraph P1 , each tethered probe further comprising an oligonucleotide having a 5' end and a 3' end, wherein the respective first fluorophore is bound to the oligonucleotide at or near the 5' end, the respective second fluorophore is bound to the oligonucleotide at or near the 3' end, and the respective quencher is bound to the oligonucleotide between the 5' and 3' ends.

P3. The tethered probe of paragraph P2, wherein the oligonucleotide is at least 15 bases long, and wherein the first fluorophore is at least 15 bases away from the second fluorophore.

P4. The tethered probe of paragraph P2, wherein the quencher is at least 7 bases away from the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end.

P5. The tethered probe of any of paragraphs P2 to P4, wherein the quencher is closer to the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end, than to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end.

P6. The tethered probe of any of paragraphs P2 to P5, wherein the respective first fluorophore and second fluorophore are bound to the oligonucleotide within about 0-10 bases from the 5’ end and 3’ end, respectively.

P7. The tethered probe of paragraph P2, wherein the oligonucleotide comprising a guanine base next to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end.

P8. The tethered probe of paragraph P1 , wherein the quencher is a third fluorophore which serves to quench a level of fluorescence from at least one of the first fluorophore and the second fluorophore.

P9. The tethered probe of any of paragraphs P1 to P8, wherein the at least one of the first fluorophore and the second fluorophore is a large stokes-shift fluorophore.

P10. The tethered probe of paragraph P1 , the tethered probe being a linear probe, wherein the respective quencher is disposed between the respective first and second fluorophores.

P11 . The tethered probe of paragraph P1 , the quencher being a first quencher, further comprising a second quencher, wherein the first and second fluorophores are bound to a first oligonucleotide, and wherein the first and second quenchers are bound to a second oligonucleotide, and wherein a sequence of the first oligonucleotide and a sequence of the second oligonucleotide are at least substantially complementary, allowing the oligonucleotides to base pair and form a duplex. P12. The tethered probe of paragraph P11 , wherein the first fluorophore and the first quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

P13. The tethered probe of paragraph P12, wherein the second fluorophore and the second quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

P14. The tethered probe of any of paragraphs P11 to P13, wherein the first and second quenchers are different.

P15. The method of any of paragraphs P1 to P14, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.

PX1. A tethered probe for multiplexed nucleic acid amplification assay comprising (1 ) an oligonucleotide with 3’ and 5’ ends; (2) a first fluorophore bound to the oligonucleotide at or near the 5’ end and a second fluorophore bound to the oligonucleotide at or near the 3’ end; and (3) at least a quencher bound to the oligonucleotide between the first fluorophore and the second fluorophore; wherein the tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first fluorophore and the second fluorophore is altered when probe is intact and cleaved.

PX2. The tethered probe of paragraph PX1 , wherein the oligonucleotide is at least 15 bases long, and wherein the first fluorophore is at least 15 bases away from the second fluorophore.

PX3. The tethered probe of paragraph PX1 , wherein the quencher is at least 7 bases away from the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end.

PX4. The tethered probe of paragraph PX3, wherein the quencher is closer to the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end, than to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end.

PX5. The tethered probe of paragraph PX1 , wherein the oligonucleotide comprises a guanine (G) base or an adenine (A) base next to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end. PX6. The tethered probe of paragraph PX1 , wherein the respective first fluorophore and second fluorophore are bound to the oligonucleotide within about CI- 10 bases from the 5’ end and 3’ end, respectively.

PX7. The tethered probe of paragraph PX1 , wherein the quencher is a third fluorophore which serves to quench a level of fluorescence from at least one of the first fluorophore and the second fluorophore.

PX8. The tethered probe of any of paragraphs PX1 to PX7, wherein the at least one of the first fluorophore and the second fluorophore is a long stokes-shift fluorophore.

PX9. The method of any of paragraphs PX1 to PX8, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.

V. Advantages and Benefits

[0137] 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.

[0138] The tethered 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 tethered probes may optionally be used with quencher probes to achieve even greater multiplexing. Significantly, the increased multiplexing provided by tethered 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.

[0139] The associated assays also have advantages and benefits. For example, the assays may be performed with larger target counts without sample splitting, simplifying workflow and maintaining the same sensitivity (e.g., LoD) 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 tethered probe assays can be generated in a familiar format, such as that used in standard quencher-probe assays, for interpretation by end users. In addition, tethered probe assays may achieve faster results by avoiding preamplification and/or melting-curve analysis. They may also reduce costs by using conventional PCR tubes/plates, without microfluidic cards or other expensive consumables.

[0140] 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.

[0141] The stoichiometry of tethered probes may have benefits for their manufacture and the subsequent analysis of tethered probe signals. For example, manufacturing quality control (QC) may be easier because the two (or more) fluorophores are molecular tethered with a fixed (e.g., 1 :1 , 1 :2, etc.) ratio. In addition, because the two fluorophores are molecularly linked or tethered at a fixed (e.g., 1 :1 ) ratio in a probe, the A Rn ratios (and other ratios) of the two fluorophores in a probe may be relatively constant. This unique feature, as noted elsewhere in this disclosure, allows accurate decoding based on both fluorophore combinatorics and the unique A Rn ratios.

VI. Conclusion

[0142] 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) a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second fluorophores can be used to assess the degree of amplification of the respective target; amplifying the plurality of targets in the mixture; measuring the fluorescence from the tethered 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 , each tethered probe further including an oligonucleotide having a 5' end and a 3' end, wherein the respective first fluorophore is bound to the oligonucleotide at or near the 5' end, the respective second fluorophore is bound to the oligonucleotide at or near the 3' end, and the respective quencher is bound to the oligonucleotide between the 5' and 3' ends.

3. The method of claim 2, wherein the quencher is closer to the 5’ end than to the 3’ end.

4. The method of claim 2, wherein the respective first fluorophore and second fluorophore are bound to the oligonucleotide within about 0-10 bases from the 5' end and 3' end, respectively.

5. The method of claim 2, wherein the oligonucleotide is hydrolyzed during amplification, such that at least one of the first fluorophore and second fluorophore is separated from the respective quencher, increasing fluorescence from each separated fluorophore.

6. The method of claim 2, wherein a same type of fluorophore is used on two distinct tethered probes, each distinct tethered probe being specific to a distinct target.

7. The method of claim 6, wherein the intensity of fluorescence from the same type of fluorophore is different from one of the two distinct probes than from the other.

8. The method of claim 6, wherein the same type of fluorophore is nearer a same end of the two distinct tethered probes, and wherein a guanine (G) base or an adenine (A) base is adjacent the same fluorophore on one of the two distinct tethered probes but not the other.

9. The method of claim 1 , wherein at least one of the first fluorophore and the second fluorophore associated with at least one tethered probe is a large stokes- shift fluorophore.

10. The method of claim 1 , wherein the nucleic acid targets are intermediate products during protein identification and/or quantification, the presence and/or level of the nucleic acid targets representing the presence and/or level of respective protein targets.

11. The method of claim 1 , the quencher being a first quencher, further comprising a second quencher, wherein the first and second fluorophores are bound to a first oligonucleotide, and wherein the first and second quenchers are bound to a second oligonucleotide, and wherein a sequence of the first oligonucleotide and a sequence of the second oligonucleotide are at least substantially complementary, allowing the oligonucleotides to base pair and form a duplex.

12. The method of claim 11 , wherein the first fluorophore and the first quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

13. The method of claim 12, wherein the second fluorophore and the second quencher are bound to, or bound next to, bases on the respective oligonucleotides that base pair when a duplex is formed.

14. The method of claim 11 , wherein the first and second quenchers are different.

15. The method of claim 1 , wherein the number of tethered probes exceeds the number of distinct fluorophores used in their construction.

16. The method of claim 1 , further comprising an additional fluorophore used only once and not present on a tethered probe.

17. The method of claim 1 , wherein at least one of the tethered probes includes a third fluorophore.

18. The method of claim 1 , wherein the tethered probe further includes a second quencher.

19. The method of claim 1 , the mixture including at least one additional nucleic acid target different from each target in the plurality of distinct nucleic acid targets, further comprising: providing a quencher probe specific to each additional target, wherein each quencher probe includes a different 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 respective additional target and higher after amplification of the respective additional target, so that changes in fluorescence from the quenchable fluorophore can be used to assess the degree of amplification of the respective additional target; amplifying each additional target in the mixture; measuring fluorescence from each respective quencher probe; and determining from the measured fluorescence a quantity representative of a level of each additional target.

20. The method of claim 19, wherein the quenchable fluorophores are selected at least in part from the group of fluorophores used to construct the tethered probes.

21. The method of claim 19, wherein the quenchers used to construct the quenchable probes are selected at least in part from the group of quenchers used to construct the tethered probes.

22. The method of claim 19, wherein the steps of amplifying the plurality of distinct targets and amplifying each additional target are performed simultaneously.

23. The method of claim 1 , the mixture including at least one additional nucleic acid target different from each target in the plurality of distinct nucleic acid targets, further comprising: providing an energy-transfer probe specific to each additional target, wherein each energy-transfer probe includes a respective donor fluorophore and a respective acceptor fluorophore, wherein the 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 additional target; amplifying each additional target in the mixture; measuring fluorescence from each respective energy-transfer probe; and determining from the measured fluorescence a quantity representative of a level of each additional target.

24. The method of claim 23, wherein the donor and acceptor fluorophores are selected from the group of fluorophores used to construct the tethered probes.

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

26. The method of claim 1, wherein the targets are identified by both the colors and intensities of the fluorescence from the respective tethered probes when the tethered probes for more than one target share a same fluorophore.

27. The method of claim 26, wherein the targets are identified by a synchronized change in fluorescence intensity at each amplification cycle.

28. The method of claim 27, wherein the synchronized change involves at least one of Ct, a signal inflection point, a dR ratio, and a AR ratio.

29. 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.

30. 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.

31 . 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.

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. 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) a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second 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 the fluorescence from the tethered probe for each target in each partition; and determining from the measured fluorescence a quantity representative of a level of each of the targets in the sample.

34. The method of claim 33, 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.

35. A kit for performing a multiplexed assay for a plurality of nucleic acid targets, comprising a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second fluorophores can be used to assess the degree of amplification of the respective target.

36. The kit of claim 35 further comprising amplification reagents sufficient for amplification of the targets.

37. The kit of claim 36, 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.

38. The kit of claim 35, further comprising at least one quencher probe.

39. The kit of claim 35, further comprising at least one energy-transfer probe.

40. 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, and a tethered probe specific to each target, wherein each tethered probe includes a first fluorophore, a second fluorophore, and a quencher, the first and second fluorophores on a given tethered probe having distinguishable excitation spectra and/or distinguishable emission spectra, wherein each tethered probe is configured such that an intensity of fluorescence emitted by at least of the first and second fluorophores is altered by amplification of the respective target, so that changes in fluorescence from the first and second fluorophores can be used to assess the degree of amplification of the respective target.

41 . The system of claim 40, further comprising one or more quencher and/or energy-transfer probes.

42. A tethered probe for multiplexed nucleic acid amplification assay comprising: a first fluorophore; a second fluorophore, having distinguishable excitation spectra and/or distinguishable emission spectra with the first fluorophore; and a quencher capable to quench a level of fluorescence from the first fluorophore and/or the second fluorophore when probe is intact; wherein the tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first fluorophore and the second fluorophore is altered between when the probe is intact and cleaved.

43. A tethered probe for multiplexed nucleic acid amplification assay comprising: an oligonucleotide with 3’ and 5’ ends; a first fluorophore bound to the oligonucleotide at or near the 5’ end and a second fluorophore bound to the oligonucleotide at or near the 3’ end; and at least a quencher bound to the oligonucleotide between the first fluorophore and the second fluorophore; wherein the tethered probe is configured such that an intensity of fluorescence emitted by at least one of the first fluorophore and the second fluorophore is altered when probe is intact and cleaved.

44. The tethered probe of claim 43, wherein the oligonucleotide is at least 15 bases long, and wherein the first fluorophore is at least 15 bases away from the second fluorophore.

45. The tethered probe of claim 43, wherein the quencher is at least 7 bases away from the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end.

46. The tethered probe of claim 45, wherein the quencher is closer to the first fluorophore, which is bound to the oligonucleotide at or near the 5’ end, than to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end.

47. The tethered probe of claim 43, wherein the oligonucleotide comprises a guanine (G) base or an adenine (A) base next to the second fluorophore, which is bound to the oligonucleotide at or near the 3’ end.

48. The tethered probe of claim 43, wherein the respective first fluorophore and second fluorophore are bound to the oligonucleotide within about 0-10 bases from the 5’ end and 3’ end, respectively.

49. The tethered probe of claim 43, wherein the quencher is a third fluorophore which serves to quench a level of fluorescence from at least one of the first fluorophore and the second fluorophore.

50. The tethered probe of claim 43, wherein the at least one of the first fluorophore and the second fluorophore is a long stokes-shift fluorophore.

51 . The method of claim 43, wherein each tethered probe is configured such that an intensity of fluorescence emitted by each of the first and second fluorophores is altered by amplification of the respective target.