Higher-order multiplexing amplification

Abstract

Described herein are compositions, methods, and systems for the detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with QSY2™ probes. Some embodiments relate to detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with a combination of QSY2™ probes and QSY™ probes, or a combination of QSY2™ probes with MGB probes, in a single reaction. Other embodiments relate to detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with a combination of QSY2™ probes with QSY™ probes, or a combination of QSY2™ probes and MGB probes in a single reaction.

Description

HIGHER-ORDER MULTIPLEXING AMPLIFICATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 570,766 filed on March 27, 2024, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates to higher-order multiplexing amplification. Described herein are compositions, methods, and systems for the detection and / or quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with fluorescein analog or derivative- based probes. BACKGROUND

[0003] Nucleic acid detection and amplification methods, such as in real-time polymerase chain reactions, use labeled probes (e.g., dual-labeled probes) to detect and / or quantify target nucleic acids like specific gene sequences or expressed messenger RNA sequences. Fluorogenic probes for use in such methods can be labeled with both a reporter and a quencher moiety. In such cases, fluorescence from the reporter is unquenched when the two dyes are physically separated via hybridization of the probe to a nucleic acid template and / or via exonuclease activity which removes one of the quencher or reporter dye components from the probe.

[0004] Real-time systems for quantitative PCR (qPCR) were improved by probe-based (rather than intercalator-based) PCR product detection. One probe-based method for detection of amplification product without separation from the primers is the 5′-exonuclease PCR assay (also referred to as the TaqMan™ assay or hydrolysis probe assay). This alternative method provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe, often referred to as a “TaqMan probe,” to its target sequence generates a substrate that is cleaved by the 5′-exonuclease activity of a DNA polymerase, such as Taq polymerase, when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified.

[0005] In general, the TaqMan probe is a non-extendable oligonucleotide attached to a fluorescent reporter dye (i.e., fluorophore) and a quencher moiety. When the TaqMan probe isintact, the reporter and quencher moieties are in close proximity, such that the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET). Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5′-end and the quencher at the 3′-end.

[0006] During the extension phase of PCR, if the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5′-exonuclease activity of a DNA polymerase possessing such activity, such as Taq polymerase, as this primer is extended. The cleavage of the probe separates the reporter dye from quencher moiety, increasing the reporter dye signal. Cleavage further removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process. Additional reporter dye molecules are cleaved from their respective probes with each cycle, affecting an increase in fluorescence intensity proportional to the amount of amplicon produced.

[0007] The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate a signal. Another advantage of fluorogenic probes is that they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters, amplification of multiple distinct sequences can be detected in a single PCR reaction, often referred to as a multiplex assay.

[0008] Current analyses of cell and tissue functionality often require extracting as much information as possible from materials that are often limited. For example, samples such as tumor biopsies are difficult to collect and usually yield only a small amount of usable nucleic acid. PCR detection and measurement of a single target analyte, referred to as a singleplex assay, has been the gold standard for analyzing clinical research samples on the nucleic acid level, and has been invaluable in extending the limits of biological knowledge for more than a quarter century.

[0009] However, the limited amount of nucleic acid obtained from specimens often forces choices to be made about how best to utilize these precious samples. Furthermore, if the sample is limited, the number of loci that can be analyzed is also limited, reducing the amount of information that can be extracted from the sample. Finally, the additional time and materials required to set up multiple single-assay reactions could increase the expense of a complex project significantly.

[0010] Multiplex PCR analysis of nucleic acids, a strategy where more than one target is amplified and quantified from a single sample aliquot, is an attractive solution to these problems.In multiplex PCR, a sample aliquot is queried with multiple probes that contain fluorescent dyes in a single PCR reaction. This increases the amount of information that can be extracted from that sample. With multiplex PCR, significant savings in sample and materials can be realized. To increase the utility of this method, multiplexed PCR using several pairs of gene-specific primers and probes to amplify and measure multiple target sequences simultaneously have been developed. Multiplexing PCR provides the following advantages. (1) Efficiency: multiplexed PCR helps conserve sample material and avoid well-to-well variation by combining several PCR assays into a single reaction. Multiplexing makes more efficient use of limited samples, such as those harboring a rare target that cannot be split into multiple aliquots without compromising the sensitivity. (2) Economy: even though the targets are amplified in unison, each one is detected independently by using a gene-specific probe with a unique reporter dye to distinguish the amplifications based on their fluorescent signal. Once optimized, a multiplexed assay is more cost effective than the same assays amplified independently.

[0011] However, currently there are limitations to the number of targets that can be analyzed in a single multiplex PCR assay. The experimental design for multiplex PCR is more complicated than for single reactions. The probes used to detect individual targets must contain unique reporter dyes with distinct spectra. The settings for excitation and emission filters of real- time detection systems vary from manufacturer to manufacturer; therefore, instruments must be calibrated for each dye as part of the experiment optimization process. Thus, one limitation in the development of multiplex PCR assays is the number of fluorophores, and hence probes, that can be effectively measured in a single reaction. For example, in multiplexed PCR, signal crosstalk between different fluorescence reporters can compromise quantification or cause false positives. It is therefore essential to select fluorophores with minimal spectral overlap. Additionally, the fluorophores, and specifically, their emission and excitation spectra, must also be compatible with the PCR instrument to be used, and specifically, the band-pass specifications for each filter-set.

[0012] Additionally, it is also important to minimize signal cross-talk by using probes that quench well. When designing a fluorescent probe, it is necessary to ensure that the fluorophore and quencher pair is compatible, given the type of detection chemistry. In addition, when designing multiplexed reactions, the spectral overlap between the fluorophores and quenchers for the different targets should be minimized to avoid possible cross-talk issues.

[0013] In general, multiplex PCR reactions have been limited to 4 probe combinations where for duplex reactions the most popular combination is FAMTMand HEXTM(JOETM / VICTM); for triplex, FAM™, HEX™ (JOE™ / VIC™), and Cyanine 5Mor NEDTM, FAM™, and VIC™; and for quadriplex, FAM™, HEX™ (JOE™ / VIC™), Texas RedTM, and Cyanine 5 dyes, or FAM™, VIC™,ABYTM, and JUNTM. Until now, most multiplex PCR instruments could take advantage of only four unique dye-quencher pairs. However, many of these instruments have the optical capability to perform higher levels of multiplexing, e.g., 5-plex and 6-plex PCR.

[0014] Thus, there is a need to provide additional probes comprising unique fluorophore / quencher combinations that allow for multiplex reactions which go beyond the use of only four spectral channels (i.e., 4-plex), such as for use in 5-plex and 6-plex multiplex PCR assays. SUMMARY

[0015] One embodiment described herein is a method of detecting or quantifying at least one of five or more target nucleic acid molecules in a sample, the method comprising: producing an amplicon of the at least one of five or more target nucleic acid molecules by performing an nucleic acid amplification reaction on the sample using a mixture including (i) at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, where the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and ii) at least five oligonucleotide primer pairs; and detecting a presence or absence of the amplicon, and / or quantifying an amount of the amplicon, by measuring fluorescence of the probe hybridized to the respective one of the five or more target nucleic acid molecules, wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first QSY2™ quencher.

[0016] In one aspect, the first QSY2™ quencher has general Formula I: R23R27(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3;Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT and Y is either H or a linkage to a solid

[0017] to a 5′-end of an oligonucleotide linker, and the first QSY2™ quencher is attached to a 3′-end of the oligonucleotide linker. In another aspect, the first dye is one selected from Cyanine 5 and Cyanine 5.5. In another aspect, a second probe comprises a product of conjugation of a second dye and a second QSY2™ quencher. In another aspect, the second QSY2™ quencher has general Formula I: R23R27NO N(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is and Y is either H or a linkage to a solid

[0018] In another aspect, the second dye is attached to a 5′-end of an oligonucleotide linker, and the second QSY2™ quencher is attached to a 3′-end of the oligonucleotide linker. In another aspect, the first dye is Cyanine 5.5 and the second dye is Cyanine 5. In another aspect, a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) a dye and a QSY™ quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher. In another aspect, at least one of the third probe and the fourth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher, and at least one of the fifth probe comprises a product of conjugation of a dye and a QSY™ quencher. In another aspect,a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) a dye and a QSY™ quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) a dye and a QSY™ quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher, and at least one of a fifth probe and a sixth probe each comprises a product of conjugation of a dye and a QSY™ quencher. In another aspect, the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM™, VIC™, ABY™, or JUN™. In another aspect, the nucleic acid amplification reaction is performed on the sample in a single reaction well. In another aspect, the nucleic acid amplification reaction is performed on the sample in a single assay. In another aspect, the performing a nucleic acid amplification reaction includes: forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a DNA polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

[0019] In another aspect, the first dye and / or the second dye has a general Formula II: R17O Kat R15X II)each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof;each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3; X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6–tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O–benzotriazole, –benzotriazole, –NR–L–OH, –NR–L– O–phosphoramidite, –NR–L–SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO– NHS, –NR–L–CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)–containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive.

[0020] Another embodiment described herein is a amplification probe set, comprising: at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, wherein the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first QSY2™ quencher; and at least five oligonucleotide primer pairs.

[0021] In one aspect, the first QSY2™ quencher has general Formula I: R23R27(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3;Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT and Y is either H or a linkage to a solid

[0022] to a 5′-end of an oligonucleotide linker, and the first QSY2™ quencher is attached to a 3′-end of the oligonucleotide linker. In another aspect, the first dye is one selected from Cyanine 5 and Cyanine 5.5.

[0023] In another aspect, a second probe comprises a product of conjugation of a second dye and a second QSY2™ quencher.

[0024] In another aspect, the second QSY2™ quencher has general Formula I: R23R27NO N(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is and Y is either H or a linkage to a solid

[0025] In another aspect, the second dye is attached to a 5′-end of an oligonucleotide linker, and the second QSY2™ quencher is attached to a 3′-end of the oligonucleotide linker. The amplification probe set according to any one of claims 21–27, wherein the first dye is Cyanine 5.5 and the second dye is Cyanine 5. In another aspect, a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) a dye and a QSY ™quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher. In another aspect, at least one of the third probe and the fourth probe each comprise a product of conjugation of adye and an MGB-NFQ quencher, and at least one of the fifth probe comprises a product of conjugation of a dye and a QSY™ quencher. In another aspect, a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) a dye and a QSY™ quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) a dye and a QSY™ quencher, or (ii) a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher. In another aspect, the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of a dye and an MGB-NFQ quencher, and at least one of a fifth probe and a sixth probe each comprises a product of conjugation of a dye and a QSY™ quencher. In another aspect, the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM™, VIC™, ABY™, and JUN™. In another aspect, the nucleic acid amplification reaction is performed on the sample in a single reaction well. In another aspect, the nucleic acid amplification reaction is performed on the sample in a single assay. In another aspect, the performing a nucleic acid amplification reaction includes: forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a DNA polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

[0026] In another aspect, the first dye and / or the second dye has a general Formula II: R1715O Kat R (Formula II)each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphaticwith terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof; each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3; X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6– tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O– benzotriazole, –benzotriazole, –NR–L–OH, –NR–L–O–phosphoramidite, –NR–L– SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO–NHS, –NR–L– CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)– containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive. DESCRIPTION OF THE DRAWINGS

[0027] Example embodiments will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. FIGS. 1-9B represent non- limiting, example embodiments as described herein.

[0028] FIG. 1 shows an exemplary 6-plex amplification plot according to embodiments described herein.

[0029] FIG.2A–G show 6-plex assay results for the reporter dyes and quenchers according to embodiments described herein (referenced as “Thermo Fisher”) and Competitor reporter dyes and quenchers for two target panels, A, and B. Probes and quenchers according to embodiments described herein are shown for Panel A (FIG.2A) and Panel B (FIG.2B) and Competitor reporter dyes and quenchers (FIG.2C–D, for Panels A and B, respectively).

[0030] FIG.3A–G shows amplification plots for 1 ng / rxn for 6-plex Panel A with reporter dyes and quenchers according to embodiments described herein comparing 6-plex vs. 1-plex reactions with all results (FIG.3A) and different reporter dye and quencher pairs: FAM™ – QSY™ (FIG.3B); ABY™ – QSY™ (FIG.3C); Cyanine 5 – QSY2™ (FIG.3D); VIC™ – QSY™ (FIG.3E); JUN™ – QSY™ (FIG.3F); and Cyanine 5.5 – QSY2™ (FIG.3G).

[0031] FIG.4A–G shows amplification plots for 1 ng / rxn for 6-plex Panel B with reporter dyes and quenchers according to embodiments described herein comparing 6-plex vs. 1-plex reactions with all results (FIG.4A) and different reporter dye and quencher pairs: FAM™ – QSY™ (FIG.4B); ABY™ – QSY™ (FIG.4C); Cyanine 5 – QSY2™ (FIG.4D); VIC™ – QSY™ (FIG.4E); JUN™ – QSY™ (FIG.4F); and Cyanine 5.5 – QSY2™ (FIG.4G).

[0032] FIG. 5A–G shows amplification plots for 1 ng / rxn for 6-plex Panel A with Competitor reporter dyes and quenchers comparing 6-plex vs. 1-plex reactions with all results (FIG.5A) and specific different reporter dye and quencher pairs: FAM – Iowa Black (FIG.5B); Cyanine 3 – Iowa Black (FIG.5C); Cyanine 5 – Tao-Iowa Black (FIG. 5D); HEX™ – Zen-Iowa Black (FIG.5E); ROX™ – Iowa Black (FIG.5F); and Cyanine 5.5 – Iowa Black (FIG.5G).

[0033] FIG. 6A–G shows amplification plots for 1 ng / rxn for 6-plex Panel B with Competitor reporter dyes and quenchers comparing 6-plex vs. 1-plex reactions with all results (FIG.6A) and specific different reporter dye and quencher pairs: FAM™ – Iowa Black (FIG.6B); Cyanine 3 – Iowa Black (FIG.6C); Cyanine 5 – Tao-Iowa Black (FIG. 6D); HEX™ – Zen-Iowa Black (FIG.6E); ROX™ – Iowa Black (FIG.6F); and Cyanine 5.5 – Iowa Black (FIG.6G).

[0034] FIG.7A shows Delta Avg Cq for the 6-plex Panel B assays. Results are shown in Table 3. FIG.7B shows a comparison of the Cyanine 5.5 (Dye 6) for embodiments described herein and the Competitor.

[0035] FIG. 8A shows 6-plex Panel B assays comparing 6-plex vs. 1-plex Avg. dCq Comparison. FIG. 8B shows an amplification plot for 1-plex and 6-plex reactions according to embodiments described herein (Thermo Fisher), and FIG.8C shows an amplification plot for 1- plex and 6-plex reactions with Competitor reporter dyes and quenchers.

[0036] FIG. 9A shows the efficiency and dynamic range of the 6-plex Panel B assays. FIG.9B–G shows the efficiency and dynamic range of reporter dyes and quenchers according to embodiments described herein compared that for reporter dyes and quencher by Competitor for Dyes 1–6. See Table 3.DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0038] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0039] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open-ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0040] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0041] As used herein, the term “or” can be conjunctive or disjunctive.

[0042] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0043] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0044] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “~” means “about” or “approximately.”

[0045] All ranges disclosed herein include both end points as values as well as all integers and fractions specified within the range. For example, a range of 0.1–2.0 includes 0.1, 0.2, 0.3, 0.4...2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0046] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0047] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control

[0048] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0049] Definitions of specific functional groups and chemical terms are described in more detail herein. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5th ed, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed, Cambridge University Press, Cambridge, 1987.

[0050] As used herein, the term “alkyl” refers to a straight or branched hydrocarbon radical having from 1 to 12 (e.g., C1–C12) carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, and the like.

[0051] As used herein, the term “alkenyl” refers to straight and branched hydrocarbon radicals having from 2 to 12 carbon atoms (e.g., C2–C12) and at least one double bond and includes, but is not limited to, ethenyl, 3-buten-1-yl, 2-ethenylbutyl, 3-hexen-1-yl, and the like. The term “alkenyl” includes cycloalkenyl, and heteroalkenyl in which 1 to 3 heteroatoms selected from O, S, N, or substituted nitrogen may replace carbon atoms.

[0052] As used herein, the term “alkynyl” refers to straight and branched hydrocarbon radicals having from 2 to 12 carbon atoms (e.g., C2–C12) and at least one triple bond and includes, but is not limited to, ethynyl, 3-butyn-1-yl, propynyl, 2-butyn-1-yl, 3-pentyn-1-yl, and the like.

[0053] As used herein, the term “cycloalkyl” refers to a monocyclic or polycyclic hydrocarbyl group having from 3 to 8 carbon atoms (e.g., C3–C8), for instance, cyclopropyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclobutyl, adamantyl, norpinanyl, decalinyl, norbornyl, cyclohexyl, and cyclopentyl. Such groups can be substituted with groups such as hydroxy, keto, amino, alkyl, and dialkylamino, and the like. Also included are rings in which 1 to 3 heteroatoms replace carbons. Such groups are termed “heterocyclyl,” which means a cycloalkyl group also bearing at least one heteroatom selected from O, S, N, or substituted nitrogen. Examples of such groups include, but are not limited to, oxiranyl, pyrrolidinyl, piperidyl, tetrahydropyran, and morpholine.

[0054] As used herein, the term “alkoxy” refers to a straight or branched chain alkyl groups having 1–10 carbon atoms (e.g., C2–C10) and linked through oxygen. Examples of such groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert- butoxy, pentoxy, 2-pentyloxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy. In addition, alkoxy refers to polyethers such as ‒O‒ (CH2)2‒O‒CH3, and the like.

[0055] The alkyl, alkenyl, alkoxy, and alkynyl groups described herein are optionally substituted (i.e., may be substituted, but are not necessarily substituted), preferably by 1 to 3 groups selected from NR4R5, phenyl, substituted phenyl, thio C1–C6alkyl, C1–C6alkoxy, hydroxy, carboxy, C1–C6alkoxycarbonyl, halo, nitrile, cycloalkyl, and a 5- or 6-membered carbocyclic ring or heterocyclic ring having 1 or 2 heteroatoms selected from nitrogen, substituted nitrogen, oxygen, and sulfur. “Substituted nitrogen” means nitrogen bearing C1–C6alkyl or (CH2)pPh where p is 1, 2, or 3. Perhalo and polyhalo substitution is also included.

[0056] Examples of substituted alkyl groups include, but are not limited to, 2-aminoethyl, 2-hydroxyethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl,ethoxycarbonylmethyl, 3-phenylbutyl, methanylsulfanylmethyl, methoxymethyl, 3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, 3-cyclopropylpropyl, pentafluoroethyl, 3-morpholinopropyl, piperazinylmethyl, and 2-(4-methylpiperazinyl)ethyl.

[0057] Examples of substituted alkynyl groups include, but are not limited to, 2- methoxyethynyl, 2-ethylsulfanylethynyl, 4-(1-piperazinyl)-3-(butynyl), 3-phenyl-5-hexynyl, 3- diethylamino-3-butynyl, 4-chloro-3-butynyl, 4-cyclobutyl-4-hexenyl, and the like.

[0058] Typical substituted alkoxy groups include aminomethoxy, trifluoromethoxy, 2- diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy, 6-carboxhexyloxy, and the like.

[0059] Further, examples of substituted alkyl, alkenyl, and alkynyl groups include, but are not limited to, dimethylaminomethyl, carboxymethyl, 4-dimethylamino-3-buten-1-yl, 5- ethylmethylamino-3-pentyn-1-yl, 4-morpholinobutyl, 4-tetrahydropyrinidylbutyl, 3-imidazolidin-1- ylpropyl, 4-tetrahydrothiazol-3-yl-butyl, phenylmethyl, 3-chlorophenylmethyl, and the like.

[0060] As used herein, the term “anion” means a negatively charged species such as chloride, bromide, trifluoroacetate, or triethylammonium. The term “cation” refers to a positively charged species, such as sodium, potassium, or ammonium.

[0061] As used herein, the term “acyl” refers to alkyl or aryl (Ar) group having from 1–10 carbon atoms bonded through a carbonyl group, i.e., R‒C(O)‒. For example, acyl includes, butis not limited to, a C1–C6 alkanoyl, including substituted alkanoyl, wherein the alkyl portion can besubstituted by an amine, amide, carboxylic, or heterocyclic group. Typical acyl groups include acetyl, benzoyl, and the like.

[0062] As used herein, the term “aryl” refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system where at least one of the rings in the ring system is an aromatic hydrocarbon ring and any other aromatic rings in the ring system include only hydrocarbons. In some embodiments, a monocyclic aryl group can have from 6 to 14 carbon atoms and a polycyclic aryl group can have from 8 to 14 carbon atoms (e.g., C8–C14). The aryl group can be covalently attached to the defined chemical structure at any carbon atom(s) that result in a stable structure. In some embodiments, an aryl group can have only aromatic carbocyclic rings, e.g., phenyl, 1- naphthyl, 2-naphthyl, anthracenyl, phenanthrenyl groups, and the like. In other embodiments, an aryl group can be a polycyclic ring system in which at least one aromatic carbocyclic ring is fused (i.e., having a bond in common with) to one or more cycloalkyl or cycloheteroalkyl rings. Examples of such aryl groups include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5,6-bicyclic cycloalkyl / aromatic ring system), cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl / aromatic ring system), imidazoline (i.e., a benzimidazolinyl group, which is a 5,6-bicyclic cycloheteroalkyl / aromatic ring system), andpyran (i.e., a chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl / aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like.

[0063] As used herein, the terms “halogen” or “halo” refer to fluorine, bromine, chlorine, or iodine.

[0064] As used herein, the term “haloalkyl” refers to an alkyl group having one or more halogen substituents. In some embodiments, a haloalkyl group can have 1 to 10 carbon atoms (e.g., C1–C8). Examples of haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, CH2Cl, C2Cl5, and the like. Perhaloalkyl groups, i.e., alkyl groups wherein all the hydrogen atoms are replaced with halogen atoms (e.g., CF3and C2F5), are included within the definition of “haloalkyl.” For example, a C1–10haloalkyl group can have the formula ‒CiH2i+1‒jXj, wherein X is F, Cl, Br, or I, i is an integer in the range of 1 to 10, and j is an integer in the range of 0 to 21, provided that j is less than or equal to 2i+1.

[0065] As used herein, the term “heteroaryl” refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from O, N, and S or a polycyclic ring system where at least one of the rings in the ring system is aromatic and contains at least one ring heteroatom. A heteroaryl group can have from 5 to 14 ring atoms (e.g., C5–C14), and contains 1– 6 ring heteroatoms (e.g., N, O, S, P, or the like). In some embodiments, heteroaryl groups can include monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, or non-aromatic cycloheteroalkyl rings. The heteroaryl group can be covalently attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O‒O, S‒S, or S‒O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S-dioxide). Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyrdazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like.

[0066] As used herein, the term “lower alkenyl” refers to alkenyl groups which contains 2 to 6 carbon atoms (e.g., C2–C6). An alkenyl group is a hydrocarbyl group containing at least one carbon-carbon double bond. As defined herein, it may be unsubstituted or substituted with the substituents described herein. The carbon-carbon double bonds may be between any two carbon atoms of the alkenyl group. It is preferred that it contains 1 or 2 carbon-carbon double bonds and more preferably one carbon-carbon double bond. The alkenyl group may be straight chained or branched. Examples include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2- butenyl, 2-methyl-1-propenyl, 1,3-butadienyl, and the like.

[0067] As used herein, the term “lower alkynyl” refers to an alkynyl group containing 2–6 carbon atoms (e.g., C2–C6). An alkynyl group is a hydrocarbyl group containing at least one carbon-carbon triple bond. The carbon-carbon triple bond may be between any two-carbon atom of the alkynyl group. In an embodiment, the alkynyl group contains 1 or 2 carbon-carbon triple bonds and more preferably one carbon-carbon triple bond. The alkynyl group may be straight chained or branched. Examples include but are not limited to ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl and the like.

[0068] As used herein, the term “carbalkoxy” refers to an alkoxycarbonyl group, where the attachment to the main chain is through the carbonyl group, e.g., ‒C(O)‒. Examples include but are not limited to methoxy carbonyl, ethoxy carbonyl, and the like.

[0069] As used herein, the term “oxo” refers to a double-bonded oxygen (i.e., =O). It is also to be understood that the terminology C(O) refers to a ‒C=O group, whether it be ketone, aldehyde or acid or acid derivative. Similarly, S(O) refers to a ‒S=O group.

[0070] As used herein, the term “cycloalkyl” refers to a non-aromatic carbocyclic group including cyclized alkyl, alkenyl, and alkynyl groups. A cycloalkyl group can be monocyclic (e.g., cyclohexyl) or polycyclic (e.g., containing fused, bridged, and / or spiro ring systems), wherein the carbon atoms are located inside or outside of the ring system. A cycloalkyl group can have from 3 to 14 ring atoms (e.g., from 3 to 8 carbon atoms for a monocyclic cycloalkyl group and from 7 to 14 carbon atoms for a polycyclic cycloalkyl group). Any suitable ring position of the cycloalkyl group can be covalently linked to the defined chemical structure. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcaryl, adamantyl, and spiro[4.5]decanyl groups, as well as their homologs, isomers, and the like.

[0071] As used herein, the term “heteroatom” refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, sulfur, phosphorus, and selenium.

[0072] As used herein, the term “cycloheteroalkyl” refers to a non-aromatic cycloalkyl group that contains at least one (e.g., one, two, three, four, or five) ring heteroatom selected from O, N, and S, and optionally contains one or more (e.g., one, two, or three) double or triple bonds. A cycloheteroalkyl group can have from 3 to 14 ring atoms and contains from 1 to 5 ring heteroatoms (e.g., from 3–6 ring atoms for a monocyclic cycloheteroalkyl group and from 7 to 14 ring atoms for a polycyclic cycloheteroalkyl group). The cycloheteroalkyl group can be covalently attached to the defined chemical structure at any heteroatom(s) or carbon atom(s) that results in a stable structure. One or more N or S atoms in a cycloheteroalkyl ring may be oxidized (e.g., morpholine N-oxide, thiomorpholine S-oxide, thiomorpholine S,S-dioxide). Cycloheteroalkyl groups can also contain one or more oxo groups, such as phthalimidyl, piperidinyl, oxazolidinoxyl, 2,4(1H,3H)-dioxo-pyrimidinyl, pyridin-2(1H)-onyl, and the like. Examples of cycloheteroalkyl groups include, among others, morpholinyl, thiomorpholinyl, pyranyl, imidazolidinyl, imidazolinyl, oxazolidinyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, azetidine, and the like.

[0073] Described herein are compositions, methods, and systems for the detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with QSY2™ probes. Some embodiments relate to detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with a combination of QSY2™ probes and QSY™ probes, or a combination of QSY2™ probes with MGB probes, in a single reaction. Other embodiments relate to detection and quantification of 5-plex and 6-plex multiplex (5 or 6 amplified nucleic acid targets) with a combination of QSY2™ TaqMan probes with QSY™ probes, or a combination of QSY2™ probes and MGB probes in a single reaction.

[0074] Described herein are compositions, methods, and systems for the detection of up to six targets in a single reaction utilizing QSY2™ probes as fifth and sixth channel probes.

[0075] Multiplex PCR is the simultaneous amplification of multiple targets in a single reaction tube. Different reporter dyes with distinct fluorescence spectra are used in multiplex PCR to track each individual amplification reaction. The real-time PCR instrument detects a signal from each reporter dye and determines the amount of each target. Multiplex PCR can reduce the amount of sample that is required for real-time PCR because it measures more than one target in a single reaction. It also can reduce reaction costs compared to performing 5 or 6 singleplex reactions independently.

[0076] Multiplex PCR with multiple reporter dyes in a single reaction well is suited for non- quantitative applications, such as genotyping and pathogen detection. For quantitative reactions, higher throughput formats such as 384-well plates and TaqMan™ OpenArray™ Plates (byThermo Fisher Scientific) provide an alternative spatial multiplexing. Where the sample is divided into smaller reactions, the sensitivity of the assay is typically reduced for spatial multiplexing.

[0077] Described herein are compositions, methods, and systems for multiplexing PCR that enables a 5-plex reaction or a 6-plex reaction for gene expression, genotyping analysis, and microRNA analysis.

[0078] According to embodiments, an oligonucleotide sequence, a reporter dye, and a quencher must be appropriate for the probe used in the assay. The quencher should be capable of quenching the reporter dye. The absorbance range of the quencher must overlap sufficiently with the emission range of the reporter dye. The choice of quencher can affect the Tmof the probe, and therefore its compatibility with the real-time PCR experiment. For example, a probe sequence paired with an MGB quencher has a higher Tm than the same sequence paired with a QSY™ or QSY2™ quencher. It is important to consider quencher choice when designing probes and calculating Tm.

[0079] For gene expression assays and genotyping assays, ABY™ dye, VIC™ dye, FAM™ dye and JUN™ dye can be utilized with either the MGB or the QSY™ non-fluorescent quencher.

[0080] The inventors have surprising found that utilizing the QSY2™ quencher with a Cyanine 5 dye and / or a Cyanine 5.5 dye, in combination with the ABY™ dye, VIC™ dye, FAM™ dye and / or JUN™ dye utilized with either the MGB or the QSY™ non-fluorescent quencher results in less distorted amplification data, and performed more consistently, than other fifth and sixth channel dyes.

[0081] Other dyes can function in multiplex PCR reactions, however, the dyes listed in Table 1 are preferred. Table 1. Dye Absorbance and Emission Wavelengths Dye Absorbance Wavelength (nm) Emission Wavelength (nm) FAM 496 520 VIC 532 552 ABY 568 583 JUN 606 618 Cyanine 5 649 667 Cyanine 5.5 678 695

[0082] FIG.1 is a 6-plex amplification plot according to embodiments described herein.

[0083] These probe set disclosed here are optimized to work together for performing multiplex PCR on real-time systems for quantitative PCR, such as QuantStudio™ Real‑Time PCRSystems (QuantStudio™ 6 / QuantStudio™ 7 Flex System, QuantStudio™ 6 Pro and 7 Pro Real- Time PCR Systems, and QuantStudio™ 12K Flex Real-Time PCR System), ViiA™ 7 Real-Time PCR Systems and 7500 / 7500 Fast Real-Time PCR Systems.

[0084] A probe set according to an example embodiment includes multiplexing up to six targets using FAM™-QSY™, VIC™-QSY™, ABY™-QSY™, JUN™-QSY™, Cyanine 5-QSY2™, and Cyanine 5.5-QSY2™ TaqMan probes.

[0085] A probe set according to another example embodiment includes multiplexing up to six targets using MGB-NFQ (FAM™, VIC™), QSY™ (ABY™, JUN™), and QSY2™ (Cyanine 5, Cyanine 5.5) probes.

[0086] A probe set according to yet another example embodiment includes multiplexing up to six targets using MGB-NFQ (FAM™, VIC™, ABY™, JUN™) and QSY2™ (Cyanine 5, Cyanine 5.5) probes.

[0087] Compared to other 5th and 6th channel single-quenched probes, QSY2 probes tend to have lower baseline signal and higher dRn. As a result, when run in combination with other probes from channels 1–4, such as FAM™-QSY™, VIC™-QSY™, ABY™-QSY™ and JUN™-QSY™ probes, QSY2™ probes report less distorted amplification results and are able retain singleplex to 6plex performance more consistently.

[0088] MGB-NFQ quenchers allows for shorter probes that are more specific and can be multiplexed up to 4 targets. QSY™ quenchers can be used for multiplexing up to 4 targets with ABY™, JUN™, FAM™, VIC™. However, the inventors have found that QSY2™ quenchers can be used for 5th and 6th target multiplexing with Cyanine 5 and Cyanine 5.5.

[0089] It will be recognized by a person or ordinary skill in the art that the fluorescence will increase as amplification of a DNA template in the presence of fluorescent double stranded DNA binding dyes. As described herein, “quantification cycle,” abbreviated “Cq” or “Ct,” refers to the cycle number at which fluorescence first rises above the threshold level. Cq is inversely related to the number of DNA templates that exist in a sample. Thus, when a low number of DNA templates exist in a sample, the Cq value will be a larger value, indicating a larger number of amplification cycles required to amplify the DNA before the emergence of a fluorescence signal. Conversely, when a high number of DNA templates exist in a sample, the Cq value will be a small value, indicating a smaller number of amplification cycles require to amplify the DNA before the emergence of a fluorescence signal.

[0090] Competitor report dye-quencher pairs exhibit a shift in the Cq value when multiplexed (6-plex). The shift of the competitor report dye-quencher pairs requires a greater number of amplification cycles when multiplexed (6-plex) as compared to their singular use (1-plex). Unlike competitor report dye-quencher pairs, the dye-quencher pairs described herein can be multiplexed without significantly impacting the Cq value. This is advantageous because multiplexing the dye-quencher pairs described herein do not impact the efficiency of the amplification as observed with the competitor’s dye-quencher pairs. Additionally, competitor report dye-quencher pairs exhibit greater variability in the Cq values amongst the individual dye- quencher pairs in the multiplex system as compared to the dye-quencher pairs described herein. It is advantageous for there to be less variability in the Cq values amongst the individual dye- quencher pairs in a multiplex system because it may allow for a decrease in the amount of time required for amplification and detection. In circumstances where a dye-quencher pairs exhibit greater variability in the Cq value, a multiplexed system will need to be amplified through the largest Cq value of an individual dye-quencher pair to ensure detection. Prolonged amplification may negatively impact the quality of the sample and the level of detection achieved due to the formation of primer dimers. The efficiency of the dye-quencher pairs described herein is underscored by 83.3% (5 / 6) of dye-quencher pairs run in a 6-plex achieved efficiency between 90–110% whereas only 33.3% (2 / 6) of competitor dye-quencher pairs achieved efficiency between 90–110% when run in a 6-plex. In addition to efficiency, the dye-quencher pairs described herein exhibit a greater dynamic range than competitor dye-quencher pairs.

[0091] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0092] Various embodiments and aspects of the inventions described herein are summarized by the following Aspects:

[0093] Aspect 1. A method of detecting or quantifying at least one of five or more target nucleic acid molecules in a sample, the method comprising: producing an amplicon of the at least one of five or more target nucleic acid molecules by performing an nucleic acid amplification reaction on the sample using a mixture including (i) at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, where the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and ii) at least five oligonucleotide primer pairs; and detecting a presence or absence of the amplicon, and / or quantifying an amount of the amplicon, by measuring fluorescence of the probe hybridized to the respective one of the five or more target nucleic acid molecules, wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first quencher, the first quencher being a first fluorescein analog or derivative.

[0094] Aspect 2. The method of Aspect 1, wherein the first quencher has general Formula I: R23R27NN(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT is either H or a linkage to a solid support.

[0095] Aspect 3. The method according to Aspect 1 or 2, wherein the first dye is attached to a 5′-end of an oligonucleotide linker, and the first quencher is attached to a 3′-end of the oligonucleotide linker.

[0096] Aspect 4. The method according to any one of Aspects 1–3, wherein the first dye is one selected from Cyanine 5 and Cyanine 5.5.

[0097] Aspect 5. The method according to any one of Aspects 1–4, wherein a second probe comprises a product of conjugation of a second dye and a second quencher, the second quencher being a second fluorescein analog or derivative that is identical to the first fluorescein analog.

[0098] Aspect 6. The method of Aspect 5, wherein the second quencher has general Formula I: R23R27NO N(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is and Y is either H or a linkage to a solid support.

[0099] Aspect 7. The method according to any one of Aspects 1–6, wherein the second dye is attached to a 5′-end of an oligonucleotide linker, and the second quencher is attached to a 3′- end of the oligonucleotide linker.

[0100] Aspect 8. The method according to any one of Aspects 1–7, wherein the first dye is Cyanine 5.5 and the second dye is Cyanine 5.

[0101] Aspect 9. The method according to any one of Aspects 1–8, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0102] Aspect 10. The method according to any one of Aspects 1–8, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB- NFQ).

[0103] Aspect 11. The method according to any one of Aspects 1–8, wherein at least one of the third probe and the fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ), and the fifth probe comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

[0104] Aspect 12. The method according to any one of Aspects 1–8, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0105] Aspect 13. The method according to any one of Aspects 1–8, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0106] Aspect 14. The method according to any one of Aspects 1–8, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ), and at least one of a fifth probe and a sixth probe each comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

[0107] Aspect 15. The method according to any one of Aspects 1–14, wherein the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM, VIC, ABY and JUN.

[0108] Aspect 16. The method according to any one of Aspects 1–15, wherein the nucleic acid amplification reaction is performed on the sample in a single reaction well.

[0109] Aspect 17. The method according to any one of Aspects 1–16, wherein the nucleic acid amplification reaction is performed on the sample in a single assay.

[0110] Aspect 18. The method according to any one of Aspects 1–17, wherein the performing a nucleic acid amplification reaction includes: forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

[0111] Aspect 19. The method according to any one of Aspects 1–18, wherein the first dye and / or the second dye has a general Formula II: R17O Kat R15X (Formula II)each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminalSO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof; each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3; X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6–tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O–benzotriazole, –benzotriazole, –NR–L–OH, –NR–L–O–phosphoramidite, –NR–L–SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO– NHS, –NR–L–CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)–containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive.

[0112] Aspect 20. An amplification probe set, comprising: at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, wherein the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first quencher, the first quencher being a first fluorescein analog; and at least five oligonucleotide primer pairs each specific for one of the five or more target nucleic acid molecules.

[0113] Aspect 21. The amplification probe set of Aspect 20, wherein the first quencher has general Formula I: (Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is and Y is either H or a linkage to a solidsupport.

[0114] Aspect 22. The amplification probe set according to any one of Aspects 20 and 21, wherein the first dye is attached to a 5′-end of an oligonucleotide linker, and the first quencher is attached to a 3′-end of the oligonucleotide linker.

[0115] Aspect 23. The amplification probe set according to any one of Aspects 20–22, wherein the first dye is one selected from Cyanine 5 and Cyanine 5.5.

[0116] Aspect 24. The amplification probe set according to any one of Aspects 20–23, wherein a second probe comprises a product of conjugation of a second dye and a second quencher, the second quencher being a second fluorescein analog that is identical to the first fluorescein analog.

[0117] Aspect 25. The amplification probe set of Aspect 24, wherein the second quencher has general Formula I: R23R27NO N(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT H is either H or a linkage to a solid support.

[0118] Aspect 26. The amplification probe set according to any one of Aspects 20-25, wherein the second dye is attached to a 5′-end of an oligonucleotide linker, and the second quencher is attached to a 3′-end of the oligonucleotide linker.

[0119] Aspect 27. The amplification probe set according to any one of Aspects 20–26, wherein the first dye is Cyanine 5.5 and the second dye is Cyanine 5.

[0120] Aspect 28. The amplification probe set according to any one of Aspects 20–27, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluoresceinanalog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0121] Aspect 29. The amplification probe set according to any one of Aspects 20-27, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ).

[0122] Aspect 30. The amplification probe set according to any one of Aspects 20-27, wherein at least one of the third probe and the fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ), and the fifth probe comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

[0123] Aspect 31. The amplification probe set according to any one of Aspects 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0124] Aspect 32. The amplification probe set according to any one of Aspects 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

[0125] Aspect 33. The amplification probe set according to any one of Aspects 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ), and at least one of a fifth probe and a sixth probe eachcomprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs

[0126] Aspect 34. The amplification probe set according to any one of Aspects 20–33, wherein the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM, VIC, ABY, and JUN.

[0127] Aspect 35. The amplification probe set according to any one of Aspects 20–36, wherein the nucleic acid amplification reaction is performed on the sample in a single reaction well.

[0128] Aspect 36. The amplification probe set according to any one of Aspects 20–35, wherein the nucleic acid amplification reaction is performed on the sample in a single assay.

[0129] Aspect 37. The amplification probe set according to any one of Aspects 20–36, wherein the performing a nucleic acid amplification reaction includes forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a DNA polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

[0130] Aspect 38. The amplification probe set according to any one of Aspects 20–37, wherein the first dye and / or the second dye has a general Formula II: R1715O Kat R X (Formula II)each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminalSO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof; each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3;X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6–tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O–benzotriazole, –benzotriazole, –NR–L–OH, –NR–L– O–phosphoramidite, –NR–L–SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO– NHS, –NR–L–CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)–containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive. EXAMPLES

[0131] Example 1

[0132] 6-plex testing with QSY™ and QSY2™ probes where a five, 10-fold serial dilution of UHR RNA was run using TaqPath™ 1-Step Multiplex Master Mix (no ROX) on the QuantStudio™ 7 Pro 384-well qPCR instrument. Conventional Probe Set

[0133] The same set of 6-plex primers and probe sequences was also tested with a combination of IDT double-quenched (Zen or TAO-Iowa Black) and single quenched (Iowa Black) probes. Results

[0134] Higher fluorescence signal (Rn and dRn) reported at cycle 40 for QSY™ and QSY2™ probes tested. Earlier or comparable Cqs reported with QSY™ and QSY2™ probes compared to IDT probes tested.

[0135] Advantages of the disclosed probe set includes detection of up to 6 targets within a single well with reduced tendency of distorted amplification curves (higher signal below the threshold, lower dRn, and non-sigmoidal amplification curve shape) with far red reporter dyes (Cyanine 5.5 and Cyanine 5).

[0136] Embodiments also provide for spectral calibration plates including the exemplary dye combinations.

[0137] For a standard 96-well plate (0.2 mL) or a fast 96-well plate (0.1 mL), spectral calibration plate 1 includes FAM™ and VIC™ dyes, plate 2 includes ABY™ and JUN™ dyes, and plate 3 includes Cyanine 5 dyes.

[0138] For a 384-well plate, spectral calibration plate 1 includes FAM™ and VIC™ dyes, and plate 2 includes ABY™, JUN™, and Cyanine 5 dyes.

[0139] According to embodiments, the amplification mixture includes PCR reaction buffers. All of the assays are amplified in the same tube. They compete for the same reagents (dNTPs, Mg2+, and polymerase). The more targets that are assayed in a multiplex reaction, the more likely it is that there will be competition for reagents and inhibition between assays. Multiplex master mixes are used to offset the effect of competition for reagents.

[0140] It is critical to calibrate the instrument for the dyes used in the experiment and to configure the instrument with the correct dyes in each well. If dyes are not assigned to their wells or dyes that are not used are assigned, the analysis will not be valid.

[0141] For calibration, the wells in the plate are labeled with the appropriate dyes because individual dye components overlap with adjacent dye channels. Overlapping spectra from each dye in a well and across all filters generates a composite spectrum that represents a raw data fluorescent reading. The PCR instrument distinguishes the contribution of each dye to the raw spectral data collected. This process is called multicomponenting.

[0142] The PCR instrument compares the raw spectra with a set of pure dye standards contained in a calibration file.

[0143] After calibration of the instrument for each dye, the PCR instrument subtracts the background from the dye that bleeds into the other dye. For example, the spectra for VIC™ dye and FAM™ dye overlap to some extent. On an instrument calibrated for both dyes, the algorithm accounts for the overlap.

[0144] According to embodiments, a multiplex reaction can contain up to twelve primers and six probes (to produce six amplicons). The amplicons should be approximately the same size.

[0145] Optimization of the concentrations of primers and probe for each target is an important step in assembling a multiplex reaction.

[0146] In an example embodiment, the forward and reverse primers each have a concentration of about 900 nm, and / or the probe has a concentration of about 250 nM.

[0147] In another example embodiment, primer-limited assays have a final forward and reverse primer concentration of about 150 nM each with about 250 nM probe concentration.

[0148] According to embodiment, no more than 800 nM total MGB probe is added in a single reaction when multiplexing.

[0149] A sample should be assayed using decreasing amounts of primer in order to determine the optimal primer concentration for each assay.

[0150] Dye and target assignments should be selected to balance fluorescence levels in the multiplex reaction. FAM™ dye and ABY™ dye can be used for targets with low to medium levels of expression. VIC™ dye, JUN™ dye, Cyanine 5 dye, and Cyanine 5.5 dye can be used for targets with high levels of expression. Because Cyanine 5 and Cyanine 5.5 are dimmer dyes, they can be used with higher levels of expression. Example 2

[0151] Amplification and Detection of 6-plex assay using QSY™ and QSY2™ probes vs. Competitor probes

[0152] Five 10-fold serial dilution of target (100 ng to 0.01 ng / rxn) plus NTC. Samples were run using TaqPath™ 1-Step Multiplex Master Mix (No ROX™) on a QuantStudio™ 7 Pro in 384-well plate with 20 μL rxn volume, 4 replicate wells and fast cycling as shown in Table 2. Two 6-plex panels were run, A and B, with different targets as shown in Table 2 with Thermo Fisher or Competitor reporter dyes and quenchers. Table 2. Assay Reactions Thermo Fisher Competitor 6-plex Target Reporter Dye Quencher Reporter Dye Quencher ACADM FAM™ QSY™ FAM™ Zen-Iowa Black HPRT1 VIC™ QSY™ HEX™ Zen-Iowa Black ABCC2 ABY™ QSY™ CY3™ Iowa Black A CD44 JUN™ QSY™ ROX™ Iowa Black SPG7 Cyanine 5 QSY2™ Cyanine 5 TAO-Iowa Black FBN1 Cyanine 5.5 QSY2™ Cyanine 5.5 Iowa Black ABCC1 FAM™ QSY™ FAM™ Zen-Iowa Black APOE VIC™ QSY™ HEX™ Zen-Iowa Black CD44 ABY™ QSY™ Cyanine 3 Iowa Black B G6PD JUN™ QSY™ ROX™ Iowa Black ESR1 Cyanine 5 QSY2™ Cyanine 5 TAO-Iowa Black TXNDC1 Cyanine 5.5 QSY2™ Cyanine 5.5 Iowa Black

[0153] Amplification plots for 100 ng / rxn are shown in FIG. 2A–G for Thermo Fisher probes and quenchers (FIG.2A–B for Panels A and B, respectively) and Competitor reporter dyes and quenchers (FIG.2C–D, for Panels A and B, respectively).

[0154] FIG.3A–G shows amplification plots for 1 ng / rxn for 6-plex Panel A with Thermo Fisher reporter dyes and quenchers comparing 6-plex vs. 1-plex reactions. In FIG. 3B, the reporter dye is FAM™, and the quencher is QSY™. In FIG.3C, the reporter dye is ABY™, and the quencher is QSY™. In FIG.3D, the reporter dye is Cyanine 5, and the quencher is QSY2™. In FIG.3E, the reporter dye is VIC™, and the quencher is QSY™. In FIG.3F, the reporter dye is JUN™, and the quencher is QSY™. In FIG.3G, the reporter dye is Cyanine 5.5, and the quencher is QSY2™.

[0155] There was better 1-plex Cq performance retention when running the same assay under 6-plex conditions.

[0156] FIG.4A–G shows amplification plots for 1 ng / rxn for 6-plex Panel B with Thermo Fisher reporter dyes and quenchers comparing 6-plex vs. 1-plex reactions. . In FIG.4B, the reporter dye is FAM™, and the quencher is QSY™. In FIG.4C, the reporter dye is ABY™, and the quencher is QSY™. In FIG.4D, the reporter dye is Cy5™, and the quencher is QSY2™. In FIG.4E, the reporter dye is VIC™, and the quencher is QSY™. In FIG.4F, the reporter dye is JUN, and the quencher is QSY™. In FIG. 4G, the reporter dye is Cy5.5, and the quencher is QSY2™.

[0157] There was better 1-plex Cq performance retention when running the same assay under 6-plex conditions.

[0158] FIG. 5A–G shows amplification plots for 1 ng / rxn for 6-plex Panel A with Competitor reporter dyes and quenchers comparing 6-plex vs.1-plex reactions.

[0159] FIG. 6A–G shows amplification plots for 1 ng / rxn for 6-plex Panel B with Competitor reporter dyes and quenchers comparing 6-plex vs.1-plex reactions.

[0160] FIG.7A–B shows Delta Avg Cq for the 6-plex Panel B assays. Results are shown in Table 3. A positive delta Avg. Cq value indicates later Cq with Competitor probe and earlier Cq with Thermo Fisher probe. About 70% (17 / 24) of the time Thermo Fisher probes have earlier Avg. Cq compared to Competitor when run in 6-plex. Earlier Cts for Competitor Cyanine 5.5 (Dye6) probes is deceiving due to poor quenching. See FIG.7B. On average, Thermo Fisher probe Cqs are earlier by 2.7 compared to Competitor when run in 6-plex. Table 3. 6-plex B - Delta Avg Cq(Competitor – Thermo)ThermoCompetitorDye FisherSample InputDelta Avg. CqScientific (ng / rxn) (Competitor – Thermo) Dye 1 100 3.193 Dye 1 10 2.940 FAM™ FAM™ Dye 1 1 2.926 Dye 1 0.1 3.022 Dye 2 100 4.724 Dye 2 10 4.537 VIC™ HEX™ Dye 2 1 6.471 Dye 2 0.1 9.315 Dye 3 100 5.017 Dye 3 10 4.852 ABY™ Cyanine 3 Dye 3 1 6.280 Dye 3 0.1 10.462 Dye 4100 −0.503 Dye 4 10 −0.612 JUN™ ROX™ Dye 4 1 −0.587 Dye 4 0.1 −0.851 Dye 5 100 1.263 Dye 5 10 1.054 Cyanine 5 Cyanine 5 Dye 5 1 2.058 Dye 5 0.1 7.556 Dye 6 100 −0.129 Dye 6 10 −0.090 Cyanine 5.5 Cyanine 5.5 Dye 6 1 −0.050 Dye 6 0.1 7.539

[0161] FIG. 8A–C show 6-plex Panel B assays comparing 6-plex vs. 1-plex Avg. dCq Comparison. 66.7% (4 / 6) of the time, Thermo Fisher probes have smaller Cq differences between 6-plex and 1-plex compared to Competitor. FIG.8B shows Thermo Cyanine 5.5 dye results and FIG.8C shows Competitor Cyanine 5.5 dye results.

[0162] FIG.9A–G shows the efficiency and dynamic range of the 6-plex Panel B assays. 66.7% (4 / 6) probes tested in 6-plex, Thermo Fisher probes reported greater PCR efficiency compared to Competitor. 83.3% (5 / 6) Thermo Fisher probes achieved PCR efficiency between 90–110% when run in 6-plex. 33.3% (2 / 6) Competitor probes achieved PCR efficiency between 90–110% when run in 6-plex. There was greater or equivalent dynamic range with Thermo Fisher probes compared to Competitor for all probes tested in 6-plex.

[0163] Competitor report dye-quencher pairs exhibit a shift in the Cqvalue when multiplexed (6-plex). The shift of the competitor report dye-quencher pairs requires a greater number of amplification cycles when multiplexed (6-plex) as compared to their singular use (1-plex). Unlike competitor report dye-quencher pairs, the dye-quencher pairs described herein can be multiplexed without significantly impacting the Cqvalue. This is advantageous because multiplexing the dye-quencher pairs described herein do not impact the efficiency of the amplification as observed with the competitor’s dye-quencher pairs. Additionally, competitor report dye-quencher pairs exhibit greater variability in the Cqvalues amongst the individual dye- quencher pairs in the multiplex system as compared to the dye-quencher pairs described herein. It is advantageous for there to be less variability in the Cqvalues amongst the individual dye- quencher pairs in a multiplex system because it may allow for a decrease in the amount of time required for amplification and detection. In circumstances where dye-quencher pairs exhibit greater variability in the Cqvalue, a multiplexed system will need to be amplified through the largest Cqvalue of an individual dye-quencher pair to ensure detection. Prolonged amplification may negatively impact the quality of the sample and the level of detection achieved due to the formation of primer dimers. The efficiency of the dye-quencher pairs described herein is underscored by 83.3% (5 / 6) of dye-quencher pairs run in a 6 plex achieved efficiency between 90–110% whereas only 33.3% (2 / 6) of competitor dye-quencher pairs achieved efficiency between 90–110% when run in a 6-plex. In addition to efficiency, the dye-quencher pairs described herein exhibit a greater dynamic range than competitor dye-quencher pairs.

Claims

CLAIMS What is claimed:

1. A method of detecting or quantifying at least one of five or more target nucleic acid molecules in a sample, the method comprising: producing an amplicon of the at least one of five or more target nucleic acid molecules by performing an nucleic acid amplification reaction on the sample using a mixture including (i) at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, where the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and ii) at least five oligonucleotide primer pairs; and detecting a presence or absence of the amplicon, and / or quantifying an amount of the amplicon, by measuring fluorescence of the probe hybridized to the respective one of the five or more target nucleic acid molecules, wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first quencher, the first quencher being a first fluorescein analog or derivative.

2. The method of claim 1, wherein the first quencher has general Formula I: R23R27NN(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT is either H or a linkage to a solid support.

3. The method according to claim 1 or 2, wherein the first dye is attached to a 5′-end of an oligonucleotide linker, and the first quencher is attached to a 3′-end of the oligonucleotide linker.

4. The method according to any one of claims 1–3, wherein the first dye is one selected from Cyanine 5 and Cyanine 5.

5.

5. The method according to any one of claims 1–4, wherein a second probe comprises a product of conjugation of a second dye and a second quencher, the second quencher being a second fluorescein analog or derivative that is identical to the first fluorescein analog.

6. The method of claim 5, wherein the second quencher has general Formula I: R23R27NO N(Formula I)each of R23, R24, R25, R26, R27, R28, R29, and R30is the same or different and isindependently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT H is either H or a linkage to a solid support.

7. The method according to any one of claims 1–6, wherein the second dye is attached to a 5′-end of an oligonucleotide linker, and the second quencher is attached to a 3′-end of the oligonucleotide linker.

8. The method according to any one of claims 1–7, wherein the first dye is Cyanine 5.5 and the second dye is Cyanine 5.

9. The method according to any one of claims 1–8, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

10. The method according to any one of claims 1–8, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

11. The method according to any one of claims 1–8, wherein at least one of the third probe and the fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ), and the fifth probe comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

12. The method according to any one of claims 1–8, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

13. The method according to any one of claims 1–8, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

14. The method according to any one of claims 1–8, wherein:the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ), and at least one of a fifth probe and a sixth probe each comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

15. The method according to any one of claims 1–14, wherein the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM, VIC, ABY and JUN.

16. The method according to any one of claims 1–15, wherein the nucleic acid amplification reaction is performed on the sample in a single reaction well.

17. The method according to any one of claims 1–16, wherein the nucleic acid amplification reaction is performed on the sample in a single assay.

18. The method according to any one of claims 1–17, wherein the performing a nucleic acid amplification reaction includes: forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

19. The method according to any one of claims 1–18, wherein the first dye and / or the second dye has a general Formula II: R1715O Kat R (Formula II)wherein: each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof; each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3; X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6–tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O–benzotriazole, –benzotriazole, –NR–L–OH, –NR–L– O–phosphoramidite, –NR–L–SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO– NHS, –NR–L–CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)–containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive.

20. An amplification probe set, comprising: at least five probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of the five or more target nucleic acid molecules, wherein the at least five probes each undergo a detectable change in fluorescence upon amplification of the respective one of the five or more target nucleic acid molecules, and wherein a first probe of the at least five probes comprises a product of conjugation of a first dye and a first quencher, the first quencher being a first fluorescein analog; and at least five oligonucleotide primer pairs each specific for one of the five or more target nucleic acid molecules.

21. The amplification probe set of claim 20, wherein the first quencher has general Formula I:R23R27NO N(Formula I)and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT H N is either H or a linkage to a solid support.

22. The amplification probe set according to any one of claims 20 and 21, wherein the first dye is attached to a 5′-end of an oligonucleotide linker, and the first quencher is attached to a 3′- end of the oligonucleotide linker.

23. The amplification probe set according to any one of claims 20–22, wherein the first dye is one selected from Cyanine 5 and Cyanine 5.

5.

24. The amplification probe set according to any one of claims 20–23, wherein a second probe comprises a product of conjugation of a second dye and a second quencher, the second quencher being a second fluorescein analog that is identical to the first fluorescein analog.

25. The amplification probe set of claim 24, wherein the second quencher has general Formula I:R23R27NO N(Formula I)and R30is the same or different and is independently selected from either H or SO3; Z is O–R, where R is H or alkyl, or NH–L, where L is O DMT H N is either H or a linkage to a solid support.

26. The amplification probe set according to any one of claims 20-25, wherein the second dye is attached to a 5′-end of an oligonucleotide linker, and the second quencher is attached to a 3′- end of the oligonucleotide linker.

27. The amplification probe set according to any one of claims 20–26, wherein the first dye is Cyanine 5.5 and the second dye is Cyanine 5.

28. The amplification probe set according to any one of claims 20–27, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

29. The amplification probe set according to any one of claims 20-27, wherein a third probe, a fourth probe and a fifth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB- NFQ).

30. The amplification probe set according to any one of claims 20-27, wherein at least one of the third probe and the fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ), and the fifth probe comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs.

31. The amplification probe set according to any one of claims 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of (i) an additional dye that is different from all other dyes and another quencher that is a fluorescein analog or derivative that is different from the first and second fluorescein analogs, or (ii) the additional dye and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

32. The amplification probe set according to any one of claims 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and a third probe, a fourth probe, a fifth probe and a sixth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non-fluorescent quencher (MGB-NFQ).

33. The amplification probe set according to any one of claims 20–27, wherein: the mixture includes six probes each having a sequence that hybridizes, and is at least partially complementary, to a respective one of six target nucleic acid molecules, and at least one of a third probe and a fourth probe each comprise a product of conjugation of an additional dye that is different from all other dyes and a minor groove binder probe-non- fluorescent quencher (MGB-NFQ), and at least one of a fifth probe and a sixth probe each comprises a product of conjugation of a third dye that is different from all other dyes and a third quencher, the third quencher being a fluorescein analog or derivative that is different from the first and second fluorescein analogs34. The amplification probe set according to any one of claims 20–33, wherein the dye of the third probe, the dye of the fourth probe, the dye of the fifth probe and / or the dye of the sixth probe is one selected from FAM, VIC, ABY, and JUN.

35. The amplification probe set according to any one of claims 20–36, wherein the nucleic acid amplification reaction is performed on the sample in a single reaction well.

36. The amplification probe set according to any one of claims 20–35, wherein the nucleic acid amplification reaction is performed on the sample in a single assay.

37. The amplification probe set according to any one of claims 20–36, wherein the performing a nucleic acid amplification reaction includes forming a mixture of the sample, the at least five probes, and the at least five oligonucleotide primer pairs, and incubating the mixture with a DNA polymerase under conditions sufficient to amplify the at least one of five or more target nucleic acid molecules.

38. The amplification probe set according to any one of claims 20–37, wherein the first dye and / or the second dye has a general Formula II: R17O Kat R15X (Formula II)each of R4, R13, and R14, when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminalSO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, –F, –Cl, –Br, or a combination thereof; each of R15, R16, R17, R18, R19, R20, R21, and R22is the same or different and is selected from the group consisting of H and SO3; each of R2and R12is the same or different and is selected from the group consisting of H and SO3;X is selected from the group consisting of –OH, –SH, –NH2, –NH–NH2, –F, –Cl, –Br, I, – O–NHS (hydroxysuccinimidyl / sulfosuccinimidyl), –O–TFP (2,3,5,6–tetrafluorophenoxy), –O–STP (4–sulfo–2,3,5,6–tetrafluorophenoxy), –O–benzotriazole, –benzotriazole, –NR–L–OH, –NR–L– O–phosphoramidite, –NR–L–SH, –NR–L–NH2, –NR–L–NH–NH2, –NR–L–CO2H, –NR–L–CO– NHS, –NR–L–CO–STP, –NR–L–CO–TFP, –NR–L–CO–benzotriazole, –NR–L–CHO, –NR–L– maleimide, NH(CH2CH2O)zCH2CH2N3, –NR–L–NH–CO–CH2–I, and an azide (N3)–containing group, where R is –H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and / or sulfur atom; Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge of the cyanine; and m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive.

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