A universal fluorescent probe activated with RNASEH2

Abstract

Methods and compositions are provided that include primers and probes for detecting nucleic acids, the probes comprising ribonucleotides that can be cleaved by RNase H2 enzyme when the probes are annealed to the reverse complement of a universal sequence introduced into a target nucleic acid, e.g., via amplification.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 376,911, filed September 23, 2022, which is incorporated by reference for all purposes. [Background technology]

[0002] Background of the Invention Various probe formats have been used to detect amplification products in bulk or partitions (e.g., dPCR or ddPCR). The most common fluorescent probe used in qPCR (quantitative PCR) and dPCR (digital PCR) is the Taqman™ probe, which requires hydrolysis by the 5' to 3' exonuclease activity of DNA polymerase to generate a fluorescent signal during each amplification cycle. Another type of fluorescent probe is the molecular beacon, which has a stem-loop structure that increases the binding specificity of the probe to its target. The disadvantage of molecular beacons is that the fluorescent signal generated is not as strong as that using hydrolysis probes, and the probe is not cleaved throughout the PCR cycle. In the context of dPCR, where the end-point fluorescent signal is read, molecular beacons hybridize to their targets during the final step, which consists of denaturing the amplified product (e.g., at 98°C for 10 minutes) and then reducing the temperature to 4°C. The default configuration for quantifying target DNA using fluorescent hydrolysis probes is to use primer pairs and fluorescent probes specific to each target amplicon. For multiplexing, this means that each target amplicon requires a set of three components: a forward primer, a reverse primer, and a fluorescent probe. Summary of the Invention

[0003] Brief Summary of the Invention In some embodiments, a method for detecting a target nucleic acid in a sample is provided. In some embodiments, the method comprises: (a) forming a reaction mixture, said reaction mixture comprising: a sample nucleic acid; and multiple forward primers containing 3' target-specific forward sequences; a plurality of reverse primers comprising a 3' target-specific reverse sequence (wherein the forward primer or reverse primer further comprises a 5' universal sequence); a probe nucleic acid comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and quencher; DNA polymerase and; RNase H2 enzyme and and (b) annealing a forward primer to a target nucleic acid in the sample nucleic acid and extending the forward primer with a polymerase using the target nucleic acid as a template to form a first strand extension product; (c) annealing a reverse primer to the first strand extension product and extending the reverse primer with a polymerase using the first strand extension product as a template to form a second strand extension product; When the reverse primer comprises a 5' universal sequence, the first strand extension product comprises the reverse complement of the 5' universal sequence, and when the forward primer comprises a 5' universal sequence, the second strand extension product comprises the reverse complement of the 5' universal sequence. Things and; (d) annealing a probe nucleic acid to the reverse complement of the 5' universal end sequence, and using an RNase H2 enzyme to cleave the ribonucleotide-annealed probe, thereby separating the quencher from the fluorophore and generating a detectable signal indicative of the presence of the target nucleic acid; Includes.

[0004] In some embodiments, (a) through (d) occur within partitions, and the target nucleic acid is distributed among the partitions such that at least a portion of the partitions do not contain the target nucleic acid.

[0005] In some embodiments, the sample nucleic acid is cell-free DNA. In some embodiments, the sample nucleic acid is from a pregnant woman and contains maternal and fetal DNA.

[0006] In some embodiments, the partition is a droplet. In some embodiments, the partition is a microwell. In some embodiments, the microwell is sealed by a lid, a cavity, or an oil layer.

[0007] In some embodiments, the detectable signal is monitored in real time, hi some embodiments, the detectable signal is monitored at the endpoint of the method.

[0008] In some embodiments, the forward primer comprises a 5' universal sequence and the concentration of the reverse primer is greater than the concentration of the forward primer. In some embodiments, the reverse primer comprises a 5' universal sequence and the concentration of the forward primer is greater than the concentration of the forward primer.

[0009] In some embodiments, the forward primer comprises a 5' universal sequence and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid, hi some embodiments, the reverse primer comprises a 5' universal sequence and the forward primer has a 5' tail sequence that does not anneal to the target nucleic acid.

[0010] In some embodiments, the probe nucleic acid is a linear probe. In some embodiments, the linear probe comprises 80-100% of the 5' universal sequence. In some embodiments, the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, 30) nucleotides that anneal to the reverse complement of the 5' universal sequence.

[0011] In some embodiments, the forward primer comprises a 5' universal sequence, and the linear probe and the reverse complement of the 5' universal sequence on the second strand extension product form a duplex with a higher melting temperature than the duplex formed from the 5' universal sequence of the forward primer and the reverse complement of the 5' universal sequence. In some embodiments, the reverse primer comprises a 5' universal sequence, and the linear probe and the reverse complement of the 5' universal sequence on the first strand extension product form a duplex with a higher melting temperature than the duplex formed from the 5' universal sequence of the reverse primer and the reverse complement of the 5' universal sequence.

[0012] In some embodiments, the probe nucleic acid forms a stem-loop and comprises, from 5' to 3', a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the ribonucleotide is in the loop sequence, and the 5' universal sequence comprises at least a portion (or all) of the loop sequence. In some embodiments, the 5' universal sequence further comprises at least a portion (or all) of the second stem sequence. In some embodiments, the first stem sequence and the second stem sequence are each 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides in length. In some embodiments, the loop sequence is 5 to 50 (e.g., 10 to 40 or 17 to 32) nucleotides in length. In some embodiments, the 5' universal sequence comprises all of the loop sequence, all of the second stem sequence, or all of the loop sequence and the second stem sequence.

[0013] In some embodiments, the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers to allow amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

[0014] In some embodiments, the RNase H2 enzyme is a Pyrococcus abyssi RNase H2 enzyme or a variant thereof, a Pyrococcus furiosis RNase H2 enzyme or a variant thereof, a Pyrococcus horikoshii RNase H2 enzyme or a variant thereof, a Thermococcus kodakarensis RNase H2 enzyme or a variant thereof, or a Thermococcus litoralis RNase H2 enzyme or a variant thereof.

[0015] In some embodiments, the reaction mixture comprises: (a) a first set of forward and reverse primers, the first set targeting a first plurality of different target nucleic acids, and the forward or reverse primers of the first set comprising a first 5' universal sequence, and the first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the first 5' universal sequence, or both; and (b) a second set of forward and reverse primers, the second set targeting a second plurality of different target nucleic acids, and the forward or reverse primers of the second set comprising a first 5' universal sequence. and a second set of probe nucleic acids comprising a second 5' universal sequence different from the first 5' universal sequence, and the second set of probe nucleic acids comprising (i) a second fluorophore, (ii) a second quencher, and (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the second 5' universal sequence, or both, such that a first amplicon derived from the first set of forward and reverse primers and having the first 5' universal sequence can be distinguished from a second amplicon derived from the second set of forward and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0016] In some embodiments, the DNA polymerase lacks 5'-3' exonuclease activity.

[0017] Also provided are reaction mixtures. In some embodiments, the reaction mixture comprises: a plurality of forward primers comprising a 3' target-specific forward sequence; a plurality of reverse primers comprising a 3' target-specific reverse sequence, wherein the forward primer or the reverse primer further comprises a 5' universal sequence; a probe nucleic acid comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and quencher; a DNA polymerase; RNase H2 enzyme and Includes:

[0018] In some embodiments, the reaction mixture further comprises a sample nucleic acid. In some embodiments, the sample nucleic acid is cell-free DNA. In some embodiments, the sample nucleic acid is from a pregnant woman and contains maternal and fetal DNA.

[0019] In some embodiments, the reaction mixture is in the partitions, and the target nucleic acid is distributed among the partitions such that at least a portion of the partitions do not contain the target nucleic acid. In some embodiments, the partitions are droplets. In some embodiments, the partitions are microwells. In some embodiments, the microwells are sealed by a lid, a cavity, or an oil layer.

[0020] In some embodiments, the forward primer comprises a 5' universal sequence and the concentration of the reverse primer is greater than the concentration of the forward primer. In some embodiments, the reverse primer comprises a 5' universal sequence and the concentration of the forward primer is greater than the concentration of the forward primer.

[0021] In some embodiments, the forward primer comprises a 5' universal sequence and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid, hi some embodiments, the reverse primer comprises a 5' universal sequence and the forward primer has a 5' tail sequence that does not anneal to the target nucleic acid.

[0022] In some embodiments, the probe nucleic acid is a linear probe. In some embodiments, the linear probe comprises 80-100% of the 5' universal sequence. In some embodiments, the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, 30) nucleotides that anneal to the reverse complement of the 5' universal sequence.

[0023] In some embodiments, the probe nucleic acid forms a stem-loop and comprises, from 5' to 3', a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the ribonucleotide is in the loop sequence, and the 5' universal sequence comprises at least a portion of the loop sequence. In some embodiments, the 5' universal sequence further comprises at least a portion of the second stem sequence. In some embodiments, the first stem sequence and the second stem sequence are each 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides in length. In some embodiments, the loop sequence is 5 to 50 (e.g., 10 to 40 or 17 to 32) nucleotides in length. In some embodiments, the 5' universal sequence comprises all of the loop sequence, all of the second stem sequence, or all of the loop sequence and the second stem sequence.

[0024] In some embodiments, the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers to allow amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

[0025] In some embodiments, the RNase H2 enzyme is a Pyrococcus abyssi RNase H2 enzyme or a variant thereof, a Pyrococcus furiosis RNase H2 enzyme or a variant thereof, a Pyrococcus horikoshii RNase H2 enzyme or a variant thereof, a Thermococcus kodakarensis RNase H2 enzyme or a variant thereof, or a Thermococcus litoralis RNase H2 enzyme or a variant thereof.

[0026] In some embodiments, the reaction mixture comprises: (a) a first set of forward primers and reverse primers, the first set targeting a first plurality of different target nucleic acids, and the forward or reverse primers of the first set comprising a first 5' universal sequence, and the first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the first 5' universal sequence, or both; (b) a second set of forward primers and reverse primers, the second set targeting a second plurality of different target nucleic acids, and the forward primers or reverse primers of the second set comprising a second 5' universal sequence that is different from the first 5' universal sequence, and the second set of probe nucleic acids comprising (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the second 5' universal sequence, or both; This allows a first amplicon derived from a first set of forward and reverse primers and having a first 5' universal sequence to be distinguished from a second amplicon derived from a second set of forward and reverse primers and having a second 5' universal sequence based on the signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0027] In some embodiments, the DNA polymerase lacks 5'-3' exonuclease activity.

[0028] Also, (a) a first set of forward primers comprising a 3' target-specific forward sequence and a reverse primer comprising a 3' target-specific reverse sequence, wherein the forward primers or reverse primers of the first set further comprise a first 5' universal sequence, and the first set targets a first plurality of different target nucleic acids; a first set of probe nucleic acids comprising: (i) a first fluorophore; (ii) a first quencher; (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of a first 5' universal sequence, or both; and (iv) at least one ribonucleotide separating the fluorophore and quencher; (b) a second set of forward primers comprising a 3' target-specific forward sequence and a reverse primer comprising a 3' target-specific reverse sequence, wherein the forward primers or reverse primers of the second set further comprise a second 5' universal sequence, and the second set targets a second plurality of different target nucleic acids; a second set of probe nucleic acids comprising (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of a second 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and quencher; This also provides a mixture in which a first amplicon derived from a first set of forward and reverse primers and having a first 5' universal sequence can be distinguished from a second amplicon derived from a second set of forward and reverse primers and having a second 5' universal sequence based on the signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0029] In some embodiments, the mixture further comprises an RNase H2 enzyme. [Brief explanation of the drawings]

[0030] [Figure 1]Figures 1A-1D illustrate the mechanism of action of molecular beacon fluorescent probes and the differences when using molecular beacons with RNA bases in their loop sequences cleaved with RNase H2-activated fluorescent probes. (1A) During the first cycle of PCR, the 5' universal tail of the forward primer is incorporated into the amplified DNA template. (1B) During subsequent PCR cycles, a reverse strand of DNA containing the reverse complement of the universal tail sequence incorporated in step A is synthesized. (1C) A standard molecular beacon (without an RNA base) is attached to the reverse complement of the universal sequence located at the 3' end of the amplified reverse DNA strand. These bind to their targets during the annealing step and are released during the denaturation step. In the context of real-time PCR, the fluorescent signal can be monitored in real time during the annealing step of each PCR cycle. As the amount of synthesized reverse DNA strand increases, the fluorescent signal increases. In dPCR, the signal is read at the endpoint. To ensure maximum binding of the molecular beacons, a final denaturation step (i.e., 98°C for 10 minutes) followed by a temperature ramp down to 4°C at 2.5°C / sec may be added. This allows all amplified DNA molecules and molecular beacons to completely denature and find their hybridization targets during the cooling step. Maximum fluorescent signal is generated during this step and remains stable for reading at room temperature. (1D) With molecular beacons containing at least one RNA base within their loop sequence, the molecular beacons bind to their targets during the annealing step, and once the RNA:DNA hybrid structure is stabilized, RNase H2 cleaves the RNA base of the molecular beacon. This generates a stronger fluorescent signal than the binding of the molecular beacon itself alone. New molecular beacons with RNA bases can hybridize and cleave during each subsequent cycle of PCR. F = fluorophore, Q = quencher, R = RNA base. [Figure 2]Figures 2A-2C show examples of RNase H2-activatable fluorescent probe designs. (2A) The 5' universal tail of the forward primer contains the same sequence as the loop sequence of the molecular beacon. One base in the center of the molecular beacon loop is changed from DNA to RNA. During the annealing process, the loop sequence of the molecular beacon binds to the reverse complement of the universal sequence, and the RNA base is cleaved by RNase H2. (2B) The 5' universal tail of the forward primer can contain the same sequence as the loop sequence and one of the stem sequences of the molecular beacon. One base in the center of the molecular beacon loop is changed from DNA to RNA. During the annealing process, the loop sequence and one stem sequence of the molecular beacon bind to the reverse complement of the universal sequence, and the RNA base is cleaved by RNase H2. (2C) The 5' universal tail of the forward primer can contain the entire length of the linear probe sequence. One base in the center of the linear probe is changed from DNA to RNA. During the annealing step, the entire length of the linear probe binds to the reverse complement of the universal sequence, and the RNA base is cleaved by Rnase H2. F=fluorophore, Q=quencher, R=RNA base. [Figure 3] Figure 3. Differences in fluorescence separation using molecular beacon probe and linear probe configurations with RNA bases tested with and without RNase H2. [Figure 4] Figure 4 displays the 60-plex results tested on a Bio-Rad QX600 Droplet Digital PCR system comparing standard and RNase H2-cleavable molecular beacons. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally carried out according to conventional methods in the art and various general references provided throughout this document (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are well-known and commonly employed in the art.

[0032] The term "amplification" refers to any in vitro means for increasing the number of copies of a nucleic acid target sequence in a linear or exponential manner, including, but not limited to, polymerase chain reaction (PCR).

[0033] "Amplifying" refers to the process of subjecting a solution to conditions sufficient to allow amplification of a polynucleotide when all components of the reaction are intact. Components of an amplification reaction include, for example, primers, polynucleotide templates, polymerase, nucleotides, and the like. The term "amplifying" typically refers to the "exponential" increase of target nucleic acids. However, as used herein, "amplifying" can also refer to the linear increase in the number of selected target sequences of nucleic acids, such as those obtained in cycle sequencing or linear amplification. In an exemplary embodiment, amplifying refers to PCR amplification using first and second amplification primers.

[0034] The term "amplification reaction mixture" refers to an aqueous solution containing various reagents used to amplify a target nucleic acid. These include enzymes, aqueous buffers, salts, amplification primers, target nucleic acids, and nucleoside triphosphates. Amplification reaction mixtures may also include stabilizers and other additives to optimize efficiency and specificity.

[0035] "Polymerase chain reaction" or "PCR" refers to a method by which a specific segment or subsequence of a target double-stranded DNA is exponentially amplified. PCR is well known to those skilled in the art; see, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., 1990. Exemplary PCR reaction conditions typically include either two- or three-step cycles. A two-step cycle has a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step followed by a separate extension step.

[0036] "Primer" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a starting point for nucleic acid synthesis. Primers can be of various lengths and, in some embodiments, are less than 60 nucleotides in length, e.g., 12-35 nucleotides in length. The length and sequence of primers for use in PCR can be designed based on principles known to those skilled in the art, see, e.g., Innis et al., supra. Primers can be DNA, RNA, or chimeras of DNA and RNA portions. In some cases, primers can contain one or more modified or unnatural nucleotide bases. In some cases, primers are labeled.

[0037] The term "probe," as described herein, refers to a nucleic acid that changes signal status depending on whether the probe anneals to a target nucleic acid. In some embodiments, the probe nucleic acid includes a blocked 3' end that prevents a polymerase from extending the annealed probe.

[0038] "Template" or "target" refers to a polynucleotide sequence that contains a polynucleotide to be amplified, and is adjacent to one or a pair of primer hybridization sites. Thus, a "target nucleic acid" includes a target polynucleotide sequence that is adjacent to at least one hybridization site for a primer. In some cases, a "target nucleic acid" includes a target polynucleotide sequence that is adjacent to the hybridization sites of a "forward" primer and a "reverse" primer.

[0039] As used herein, "nucleic acid" refers to DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof, including, but not limited to, those that provide additional charge, polarizability, hydrogen bonding, electrostatic interactions, points of attachment to the nucleic acid ligand bases or nucleic acid ligand as a whole, and chemical groups that incorporate functionality. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) (see, e.g., WO 99 / 14226, WO 98 / 39352, WO 2003 / -20739, U.S. Pat. No. 7,053,207), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2'-sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amine, 4-thiouridine substitutions, 5-bromo- or 5-iodo-uracil substitutions, backbone modifications, methylations, unusual base pair combinations such as isobases, isocytidines, and isoguanidines, and the like. Nucleic acids can also include unnatural bases, such as, for example, nitroindoles. Modifications can also include 3' and 5' modifications, including, but not limited to, capping with fluorophores (e.g., quantum dots) or other moieties.

[0040] "Polymerase" refers to an enzyme that carries out template-directed synthesis of polynucleotides, e.g., DNA and / or RNA. The term encompasses both full-length polypeptides and domains with polymerase activity. DNA polymerases are well known to those of skill in the art and include, but are not limited to, DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to, Klenow fragment (New England Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9°N™ DNA polymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (New England Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNA polymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (New England Biolabs® Inc.). At least five families of DNA-dependent DNA polymerases are known, with most falling into families A, B, and C. Other types of DNA polymerases include phage polymerases.

[0041] As used herein, the terms "partition" or "partitioned" refer to the separation of a sample into multiple portions, or "partitions." The partitions are generally physical, such that the sample in one partition does not mix, or substantially does not mix, with the sample in an adjacent partition. The partitions can be solid or fluid. In some embodiments, the partitions are solid partitions, e.g., microchannels. In some embodiments, the partitions are fluid partitions, e.g., droplets. In some embodiments, the fluid partitions (e.g., droplets) are mixtures of immiscible fluids (e.g., water and oil). In some embodiments, the fluid partitions (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil).

[0042] Detailed Description of the Invention Methods for detecting target nucleic acids are provided using probes containing ribonucleotides. The probes are cleaved by RNase H2 enzyme when bound to a universal sequence introduced into the target nucleic acid by a primer. For example, the probes described herein can have a fluorophore moiety and a quencher moiety separated by a ribonucleotide, thereby relatively quenching the signal from the fluorophore in the intact probe compared to when the probe is cleaved at the ribonucleotide by RNase H2 enzyme. The inventors have found such probes to be of particular use in digital amplification assays and also useful in real-time bulk amplification assays. Some advantages of these methods include better separation of signal (target) from background (no target), as well as the ability to use DNA polymerases lacking 5'-3' exonuclease activity. The methods described herein can also be used in various multiplex formats, allowing for the detection of multiple different amplicons as desired. Additional advantages will become apparent from the remainder of this disclosure.

[0043] The method can involve forming a reaction mixture that includes a sample nucleic acid, a forward primer and a reverse primer, a probe as described herein, a DNA polymerase, and an RNase H2 enzyme, each of which is discussed below, followed by a discussion of how they are used.

[0044] A target nucleic acid is any DNA sequence (e.g., dsDNA, cDNA) to be detected in a sample. A biological sample can be obtained from any organism, such as an animal, a plant, a fungus, a pathogen (e.g., a bacterium or virus), or any other life form. In some embodiments, the biological sample is from an animal, such as a mammal (e.g., a human or non-human primate, cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., a chicken), or a fish. A biological sample can be any tissue or fluid obtained from a living organism, such as blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, neural tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue), cultured cells, such as primary cultures, explants, and transformed cells, stem cells, stool, urine, etc. In some embodiments, the sample is a cell-free nucleic acid sample. In some embodiments, the cell-free DNA sample is from a pregnant woman (e.g., a cell-free sample from maternal blood containing maternal and fetal DNA), or from a person who has, has had, or is suspected of having cancer, or is an organ transplant recipient.

[0045] The reaction mixture will have multiple forward primers containing one or more 3' target-specific forward sequences. The terms "forward" and "reverse" are arbitrary designations and indicate that the forward and reverse primers anneal to opposite strands of a double-stranded DNA molecule and have their 3' ends oriented toward each other so that, when annealed, an amplicon forms under PCR conditions. The 3' target-specific forward sequences can vary in composition and length and can be designed to specifically amplify a particular target nucleic acid in a mixture of different nucleic acids. In some embodiments, the 3' target-specific forward sequence is 10-30 nucleotides in length and anneals to or adjacent to the target nucleic acid, although other lengths can be used in other embodiments.

[0046] The multiple forward primers will contain numerous copies of the primer to achieve the desired amplification. In a multiplex reaction, the multiple forward primers will further include multiple different forward primers with different 3' target-specific forward sequences, allowing for amplification of different targets in the same reaction. As a simple example, a reaction can have a copy of a first forward primer with a first 3' target-specific forward sequence and a copy of a second forward primer with a second (different) 3' target-specific forward sequence, allowing for amplification of a first target and a second target, if present in the sample. As discussed further below, the number of different forward primers in the same reaction can be large, e.g., at least 2, 5, 10, 20, 30, 40, 50, 70, 100, 120, or more, e.g., from 2 to 200. As discussed below, in some embodiments, the universal sequence will be located on the forward primer, and in this case, at the 5' end of the forward primer.

[0047] In some embodiments, multiple reverse primers are provided.For example, in some embodiments, for each forward primer with unique 3' target specific forward sequence, there will be one reverse primer with unique 3' target specific reverse sequence, so that forward primer and reverse primer will amplify specific target nucleic acid together.Similar to forward primer, in multiplex option, reverse primer with different 3' target specific reverse sequence can be adopted in reaction mixture.

[0048] Either the forward primer or the reverse primer further comprises a 5' universal sequence, but in some embodiments, not both. The 5' universal sequence is a sequence common to the set of primers but does not anneal to the target sequence in the initial amplification round (they are not target-specific). See, e.g., FIG. 1. The 5' universal sequence can be of any length desired, as long as it is long enough for probe annealing during the reaction, as discussed below. In some embodiments, for example, the 5' universal sequence is 10-50 nucleotides long, e.g., 15-40, 15-35, or 20-35 nucleotides long. At least a portion, and in some embodiments, all, of the 5' universal primer and at least a portion of the probe sequence, including ribonucleotides of the probe, will have identical sequences to each other, thereby allowing probes containing ribonucleotides and reverse complements of the universal sequence to anneal and be cleaved at the ribonucleotides by RNase H2 enzyme, as further described below.

[0049] In embodiments in which pairs of forward and reverse primers are provided, it may be advantageous to include a larger number (e.g., a higher concentration) of primers that do not have a 5' universal sequence compared to primers that have a 5' universal sequence. For example, in embodiments in which the forward primer has a 5' universal sequence, in some cases the concentration of the reverse primer will be higher than the concentration of the forward primer (e.g., at least 1.5-fold, 2-fold, 3-fold, 5-fold, or 10-fold higher). In embodiments in which the reverse primer has a 5' universal sequence, in some cases the concentration of the forward primer will be higher than the concentration of the reverse primer (e.g., at least 1.5-fold, 2-fold, 3-fold, 5-fold, or 10-fold higher). As an example, in some embodiments, primers lacking a 5' universal sequence are provided at a concentration of 500 to 2000 nM (e.g., 900 or 1000 nM), and primers with a 5' universal sequence are provided at a concentration of 50 to 200 nM (e.g., 90 or 100 nM). This is useful for generating an excess of the strand that has the reverse complement of the 5' universal sequence that is detected by the probe.

[0050] In some multiplexing embodiments, some primers can have different 5' universal sequences, allowing for the use of probes of different "colors" in the same reaction. For example, in some embodiments, a first set of primers (forward or reverse) has a first 5' universal sequence, and a second set of primers (forward or reverse) has a second 5' universal sequence. In these embodiments, the reaction mixture contains a first probe with a sequence identical to part or all of the first 5' universal sequence and a second probe with a sequence identical to part or all of the second 5' universal sequence. If the first and second probes have different linked fluorophores or quenchers such that the emitted signals have different wavelengths (colors), the first and second probe signals can be distinguished. While the above examples are illustrated using probes associated with two 5' universal sequences, probes associated with more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, etc.) 5' universal sequences can be employed, with each probe distinguishable from the others by the wavelength of its signal.

[0051] Furthermore, in some embodiments, each set of primers with the same 5' universal sequence can contain primers with two or more different 3' target-specific forward (or reverse, depending on the primer) sequences. Thus, for example, a first set of forward primers can contain two, five, ten, twenty, or more different 3' target-specific forward sequences, each with a first 5' universal sequence and therefore detectable by a first probe, and a second set of forward primers can contain two, five, ten, twenty, or more different 3' target-specific forward sequences, each with a second 5' universal sequence and therefore detectable by a second probe. This allows for the monitoring of a very large number of different amplifications, all contained in a single reaction mixture. For example, the inventors used six distinct probes to probe twenty different amplification reactions, each with its own 5' universal sequence, allowing for the monitoring of 120 different reactions in digital format.

[0052] Probe nucleic acids use energy transfer between two moieties, for example, a donor fluorophore and an acceptor moiety separated by a ribonucleotide. In some embodiments, the probe nucleic acid comprises a fluorophore moiety and a quencher moiety (i.e., an acceptor that absorbs the energy emitted by the donor fluorophore but does not subsequently fluoresce itself) separated by a ribonucleotide, such that upon cleavage of the ribonucleotide, the two moieties are separated, resulting in a detectable signal. The fluorophore and quencher can be located at the end of the probe, or one or both can be linked to an internal nucleotide within the probe. In some embodiments, the proximity of the quencher to the fluorophore in an intact probe results in a quenched fluorescent signal, while a cleaved probe results in an increased detectable signal due to reduced or absent quenching. In some embodiments in which two or more different probes are used, the different probes will have different fluorophores or quenchers, or both, such that the different probes emit signals at different wavelengths that can be distinguished from each other by the sensor. Some quenchers are capable of quenching a wide range of wavelengths, so in some embodiments the same quencher is used for two or more different probes, each of which has a different fluorophore that emits at a different wavelength.

[0053] Fluorescent agents can include various organic and / or inorganic small molecules, or various fluorescent proteins and their derivatives. Numerous fluorophores and quenchers have been reported in the literature and are therefore known to those skilled in the art, and many are readily available from commercial suppliers to the biotechnology industry. Literature sources for fluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Dexter, DL, J. of Chemical Physics 21:836-850 (1953); Hochstrasser et al., Biophysical Chemistry 45:133-141 (1992); Selvin, P., Methods in Enzymology 246:300-334 (1995); Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann. Rev. Biochem., 47:819-846 (1978); Wang et al., Tetrahedron Letters 31:6493-6496 (1990); Wang et al., Anal. Chem. 67:1197-1203 (1995). Non-limiting examples of fluorophores include cyanines, fluoresceins (e.g., 5'-carboxyfluorescein (FAM), Oregon Green, and Alexa 488), HEX, rhodamines (e.g., N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)), eosin, coumarins, pyrenes, tetrapyrroles, arylmethines, oxazines, polymer dots, and quantum dots. In some embodiments, the fluorophore is selected from HEX, FAM, Cy5, Cy5.5, ROX, Atto 590, Alexa 405, Pacific Blue, ABY, and Texas Red.Exemplary quenchers can include, but are not limited to, Iowa Black® FQ, Iowa Black® RQ, Black Hole Quencher®-1, Black Hole Quencher®-2, DABCYL, and ZEN internal quencher.

[0054] The nucleotide sequence of the probe (including ribonucleotide positions) will contain at least 25%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the 5' universal sequence. For example, in some embodiments, the probe can contain 10-50, e.g., 15-30, nucleotides identical to the 5' universal sequence. At the aligned positions, the ribonucleotides will contain the same nucleotides (but in ribonucleotide form) as the 5' universal sequence, except that if a position in the 5' universal sequence is thymine, the ribonucleotide will be its RNA equivalent, i.e., uracil. The nucleotide sequence will be composed of non-ribonucleotides, except for one or more ribonucleotides separating the fluorophore and quencher moieties. The non-ribonucleotides can be, for example, deoxyribonucleotides (those that form DNA) or analogs thereof. In some embodiments, the probe can contain one or more non-natural nucleotides or analogs thereof (e.g., peptide nucleic acid (PNA) or locked nucleic acid (LNA)), or other nucleotides that have a higher affinity for the reverse complement of the 5' universal sequence compared to a primer having a 5' universal sequence (composed of DNA). The increased affinity (higher melting temperature (Tm)) of the probe for the reverse complement of the 5' universal sequence compared to a primer having a 5' universal sequence for the reverse complement of the 5' universal sequence can be used to further improve signal-to-noise differentiation.

[0055] As described above, one or more ribonucleotides are positioned within the probe between the fluorophore and quencher, such that when the RNase H2 enzyme cleaves at the ribonucleotide, the fluorophore and quencher diffuse away from each other to generate a detectable signal. Furthermore, the RNase H2 enzyme cleaves ribonucleotides annealed to their deoxyribonucleotide counterparts in the context of two annealed strands (in this case, the probe and the reverse complementary strand of the 5' universal sequence). RNase H2 enzyme cleavage is more efficient when there are multiple annealed nucleotides (e.g., 2, 3, 4, 5, 6, or more) on either side of the ribonucleotide. Therefore, in some embodiments, the ribonucleotide is positioned in the middle section of the probe or at least 2, 3, 4, 5, 6, or more nucleotides away from either end of the probe.

[0056] Optionally, the probe is modified at the 3' end so that polymerase cannot extend the probe. In some embodiments, a fluorophore or quencher is linked to the 3' end of the probe, which blocks polymerase from extending the probe. In other embodiments, the 3' end of the probe can be modified to block extension. Non-limiting modifications of the 3' end can include, for example, 3' inverted dT, 3' C3 spacer, 3' amino, or 3' phosphorylation.

[0057] Various probe formats can be used. In some embodiments, the probe is a linear probe, meaning that the sequence within the probe does not form a stem-loop structure or otherwise self-anneal. In other embodiments, the probe can be in a stem-loop format, where the ribonucleotide is located within the loop portion. See, for example, Figure 2. The probe concentration can be selected to avoid background while still producing a detectable signal. For example, in some embodiments, the probe is provided at a concentration of 100-1000 nM, e.g., 500 nM. In some embodiments, the probe concentration is lower than the concentration of the limiting primer (i.e., the primer with the 5' universal sequence), and in some embodiments, is 2-10 times the concentration of the limiting primer.

[0058] The linear probe can be any length as desired. Exemplary probe nucleic acids can have, for example, at least 10 (e.g., at least 15, 20, 25, 30) consecutive or non-consecutive nucleotides that are reverse complementary to the 5' universal sequence. In some embodiments, the linear probe comprises a sequence that is at least 80%, 90%, or 100% identical to the 5' universal probe sequence, thereby improving the probe's ability to compete for the reverse complement of the universal 5' sequence compared to a primer having a 5' universal sequence.

[0059] A stem-loop probe has a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, allowing the two stem sequences to self-anneal to form a stem-loop. At least a portion of the loop sequence containing ribonucleotides has the same sequence as the 5' universal sequence. In some embodiments, at least a portion of the second stem sequence is also the same as the sequence in the 5' universal sequence. In some embodiments, the entire loop sequence or the second stem sequence, or both, are identical to the adjacent sequences of the 5' universal sequence. See, for example, Figure 2.

[0060] The lengths of the stem and loop sequences can be varied as desired. In some embodiments, the first and second stem sequences are each 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides in length, although other lengths are also possible. In some embodiments, the loop sequence is 5 to 50 (e.g., 10 to 40 or 17 to 32) nucleotides in length, although other lengths are also possible.

[0061] The reaction mixture can include a DNA polymerase that acts to extend at least one of the primers in a template-dependent manner. In some embodiments, the DNA polymerase is a thermostable polymerase. Thermostable polymerases include those from Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), Bacillus sterothermophilus (Bst), Sulfolobus acidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium DNA polymerases are isolated from a wide variety of thermophilic bacteria, including Methanobacterium thermoautotrophicum (Mth), as well as other species. DNA polymerases are known in the art and commercially available. In some embodiments, the DNA polymerase is Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, or an active mutant, variant, or derivative thereof. In some embodiments, the DNA polymerase is Taq DNA polymerase. In some embodiments, the DNA polymerase is a high-fidelity DNA polymerase (e.g., iProof™ high-fidelity DNA polymerase, Pusion® high-fidelity DNA polymerase, Q5® high-fidelity DNA polymerase, Platinum® Taq high-fidelity DNA polymerase, Accura® high-fidelity polymerase).In some embodiments, the DNA polymerase is a fast-start polymerase (e.g., FastStart™ Taq DNA polymerase or FastStart™ High-Fidelity DNA polymerase). In some embodiments, the polymerase lacks 5'-3' exonuclease activity, e.g., as found in Family B polymerases.

[0062] As described above, the present methods and compositions can involve the activity of RNase H2 enzymes. RNase H2 enzymes cleave ribonucleotides within an RNA / DNA duplex. Various RNAse H2 enzymes are known and can be used in the methods described herein. Exemplary RNAse H2 enzymes are described, for example, from Pyrococcus abyssi, Pyrococcus furiosis, Pyrococcus horikoshii, Thermococcus kodakarensis, and Thermococcus litoralis. In addition, various mutant enzymes have been developed from these native enzymes, as described, for example, in International Publication No. 2018 / 031625, which is incorporated by reference.

[0063] The methods herein involve forming a reaction mixture having some or all of the components described above, as well as reagents necessary for primer extension (eg, amplification).

[0064] The amplification reaction mixture will also contain nucleotides. Nucleotides for use in the methods described herein can be any nucleotide useful for nucleic acid polymerization. Nucleotides can be naturally occurring, unusual, modified, derivative, or artificial. Nucleotides are generally unlabeled in the embodiments described herein.

[0065] In some embodiments, the amplification reaction mixture includes one or more buffers or salts. A wide variety of buffers and salt solutions and modified buffers are known in the art. For example, in some embodiments, the buffer is TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, or CAPS. In some embodiments, the salt is potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, or lithium acetate. In some embodiments, the amplification reaction mixture includes a salt (e.g., potassium chloride) at a concentration of about 10 mM to about 100 mM.

[0066] In some embodiments, the amplification reaction mixture includes one or more stabilizers. Stabilizers for use in the methods described herein include polyols (glycerol, threitol, etc.), polyethers, including cyclic polyethers, polyethylene glycol, organic or inorganic salts (ammonium sulfate, sodium sulfate, sodium molybdate, sodium tungstate, organic sulfonates, etc.), sugars, polyhydric alcohols, amino acids, peptides, or carboxylic acids, quenchers and / or scavengers (mannitol, glycerol, reduced glutathione, superoxide dismutase, bovine serum albumin (BSA) or gelatin, spermidine, dithiothreitol (or mercaptoethanol)), and / or detergents (TRITON® X-100 [octophenol (ethylene glycol ether)], THESIT® [polyoxyethylene 9 lauryl ether (polydocanol C)], etc.). 12 E9)], TWEEN® (Polyoxyethylene sorbitan monolaurate 20, NP40) and BRIJ®-35 (Polyoxyethylene 23 lauryl ether)).

[0067] Once the reaction mixture is formed, it is subjected to conditions that allow primer extension using one or more primers that anneal to the target nucleic acid to be detected. The reaction mixture is subjected to primer extension conditions, which can be but are not limited to PCR conditions, that allow the primer, if present, to anneal to the target nucleic acid and be extended by the polymerase in a template (i.e., target nucleic acid) specific manner. The reaction can be carried out in bulk or in partitions, and the signal can be monitored at the end point or in real time, i.e., continuously or cycle by cycle.

[0068] In embodiments including a forward primer and a reverse primer, the forward primer is extended in the presence of the target nucleic acid to form a first strand, followed by extension of the reverse primer using the first strand as a template to form a second strand complementary to the first strand. Depending on whether the 5' universal sequence is on the forward primer or the reverse primer, the first strand or the second strand will contain the 5' universal strand, respectively, and therefore the second strand or the first strand will contain the reverse complement of the 5' universal sequence, respectively. Because the probe nucleic acid and RNase H2 enzyme are also present in the reaction mixture, once the reverse complement of the 5' universal sequence is generated, the probe is annealed to the reverse complement of the 5' universal sequence, and the annealed probe is cleaved at the ribonucleotide by the RNase H2 enzyme, separating the fluorophore and quencher, resulting in a detectable fluorescent signal. The amount of this signal can indicate the amount or at least the presence of the target nucleic acid in the bulk reaction, or alternatively, if the reaction is in partitions, the number of partitions having a signal above the threshold will be proportional to the amount of target nucleic acid in the sample. Generally, the amount of sample nucleic acid and the number of partitions are selected such that at least some partitions are empty, as specified by the Poisson distribution.

[0069] Methods and compositions for partitioning samples are described, for example, in published patent applications WO 2010 / 036,352, U.S. Patent Publication Nos. 2010 / 0173,394, 2011 / 0092,373, and 2011 / 0092,376, the contents of each of which are incorporated herein by reference in their entirety. The multiple mixture partitions can be in the form of multiple emulsion droplets, multiple microwells, or the like.

[0070] In some embodiments, the sample nucleic acid can be partitioned into multiple mixture partitions, and then one or more amplification primers, probes, enzymes, oligonucleotides, or combinations thereof can be introduced into the multiple mixture partitions. Methods and compositions for delivering reagents to one or more mixture partitions include microfluidic methods known in the art, droplet or microcapsule fusion, coalescence, condensation, rupture, or disintegration (e.g., as described in U.S. Patent Publication Nos. 2015 / 0027,892, 2014 / 0227,684, WO 2012 / 149,042, and WO 2014 / 028,537), droplet injection methods (e.g., as described in WO 2010 / 151,776), and combinations thereof.

[0071] The mixture partition can be a picowell, nanowell, or microwell. The mixture partition can be a pico-, nano-, or micro-reaction chamber, such as a picocapsule, nanocapsule, or microcapsule. The mixture partition can be a picochannel, nanochannel, or microchannel. The mixture partition can be a droplet, for example, an emulsion droplet.

[0072] In some embodiments, the sample nucleic acid and PCR reaction components are partitioned into multiple droplets. In some embodiments, the droplets comprise an emulsion composition, i.e., a mixture of immiscible fluids (e.g., water and oil). In some embodiments, the droplets are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil). In some embodiments, the droplets are oil droplets surrounded by an immiscible carrier fluid (e.g., aqueous solution). In some embodiments, the droplets are relatively stable and have minimal coalescence between two or more droplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the droplets generated from the sample coalesce with other droplets. Emulsions may also have limited flocculation, a process in which the dispersed phase comes out of suspension in flakes. Methods of emulsion formation are described, for example, in published patent applications WO 2011 / 109546 and WO 2012 / 061444, the entire contents of each of which are incorporated herein by reference.

[0073] In some embodiments, droplets are formed by flowing an oil phase through an aqueous sample containing the sample and reaction components. The oil phase may include a fluorinated base oil, which may be further stabilized by combination with a fluorinated surfactant such as a perfluoropolyether. In some embodiments, the base oil includes one or more of HFE 7500, FC-40, FC-43, FC-70, or another common fluorinated oil. In some embodiments, the oil phase includes an anionic fluorosurfactant. In some embodiments, the anionic fluorosurfactant is ammonium krytox (Krytox-AS), an ammonium salt of Krytox FSH, or a morpholino derivative of Krytox FSH. Krytox-AS may be present at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w / w). In some embodiments, the concentration of Krytox-AS is about 1.8%. In some embodiments, the concentration of Krytox-AS is about 1.62%. The morpholino derivative of Krytox FSH may be present at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w / w). In some embodiments, the concentration of the morpholino derivative of Krytox FSH is about 1.8%. In some embodiments, the concentration of the morpholino derivative of Krytox FSH is about 1.62%.

[0074] In some embodiments, the oil phase further comprises an additive to adjust oil properties such as vapor pressure, viscosity, or surface tension. Non-limiting examples include perfluorooctanol and 1H,1H,2H,2H-perfluorodecanol. In some embodiments, 1H,1H,2H,2H-perfluorodecanol is added to a concentration of about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0% (w / w). In some embodiments, 1H,1H,2H,2H-perfluorodecanol is added to a concentration of about 0.18% (w / w).

[0075] In some embodiments, emulsions are formulated to produce highly monodisperse droplets with a liquid-like interfacial membrane that can be converted to microcapsules with a solid-like interfacial membrane upon heating; these microcapsules may behave as bioreactors capable of retaining their contents throughout the incubation period. See, e.g., U.S. Pat. No. 10,378,048. Conversion to the microcapsule form may occur upon heating. For example, such conversion may occur at temperatures above about 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 95°C. A fluid or mineral oil cover may be used to prevent evaporation during the heating process. Excessive continuous phase oil may or may not be removed prior to heating. Biocompatible capsules may be resistant to coalescence and / or flocculation over a wide range of thermal and mechanical treatments. Following conversion, the microcapsules may be stored at about -70°C, -20°C, 0°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0076] Microcapsule partitions, which may contain one or more polynucleotide sequences and / or one or more primer sets, may be resistant to coalescence, especially at high temperatures. As a result, capsules can be incubated at very high densities (e.g., the number of partitions per unit volume). In some embodiments, more than 100,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 5,000,000, or 10,000,000 partitions per mL may be incubated. In some embodiments, incubation of the sample probe occurs within a single well, such as the well of a microtiter plate, without intermixing between partitions. The microcapsules may also contain other components necessary for incubation.

[0077] In some embodiments, the sample has at least 500 partitions, at least 1000 partitions, at least 2000 partitions, at least 3000 partitions, at least 4000 partitions, at least 5000 partitions, at least 6000 partitions, at least 7000 partitions, at least 8000 partitions, at least 10,000 partitions, at least 15,000 partitions, at least 20,000 partitions, at least 30,000 partitions, at least 40,000 partitions, at least 50,000 partitions, at least 60,000 partitions, at least 70,000 partitions, at least 80,000 partitions, at least 90,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 300,000 partitions, at least 400,000 partitions, at least 500,000 partitions partitions, at least 600,000 partitions, at least 700,000 partitions, at least 800,000 partitions, at least 900,000 partitions, at least 1,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000 partitions, at least 20,000 ,000 partitions, at least 30,000,000 partitions, at least 40,000,000 partitions, at least 50,000,000 partitions, at least 60,000,000 partitions, at least 70,000,000 partitions, at least 80,000,000 partitions, at least 90,000,000 partitions, at least 100,000,000 partitions, at least 150,000,000 partitions, at least 200,It is partitioned into 000,000 partitions.

[0078] In some embodiments, the droplets generated are substantially uniform in shape and / or size. For example, in some embodiments, the droplets have a substantially uniform average diameter. In some embodiments, the droplets generated have an average diameter of about 0.001 micrometers, about 0.005 micrometers, about 0.01 micrometers, about 0.05 micrometers, about 0.1 micrometers, about 0.5 micrometers, about 1 micrometer, about 5 micrometers, about 10 micrometers, about 20 micrometers, about 30 micrometers, about 40 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers, about 150 micrometers, about 200 micrometers, about 300 micrometers, about 400 micrometers, about 500 micrometers, about 600 micrometers, about 700 micrometers, about 800 micrometers, about 900 micrometers, or about 1000 micrometers. In some embodiments, the droplets produced have an average diameter of less than about 1000 micrometers, less than about 900 micrometers, less than about 800 micrometers, less than about 700 micrometers, less than about 600 micrometers, less than about 500 micrometers, less than about 400 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 50 micrometers, or less than about 25 micrometers. In some embodiments, the droplets produced are non-uniform in shape and / or size.

[0079] In some embodiments, the droplets generated are substantially uniform in volume, for example, in some embodiments, the droplets generated are about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, or about 2.5 nL. , about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL. In some embodiments, the droplets have an average volume of about 50 picoliters to about 2 nanoliters. In some embodiments, the droplets have an average volume of about 0.5 nanoliters to about 50 nanoliters. In some embodiments, the droplets have an average volume of about 0.5 nanoliters to about 2 nanoliters.

[0080] In some embodiments, the amplification reaction is a droplet digital PCR reaction. Methods for performing PCR in droplets are described, for example, in U.S. Patent Publication Nos. 2014 / 0162266, 2014 / 0302503, and 2015 / 0031034, the contents of each of which are incorporated by reference. In some embodiments, a QX200, QX600, or QX One Droplet Digital PCR (ddPCR) system (Bio-Rad) is used.

[0081] In some embodiments, the detection reagent or detectable label in the partition can be detected using any of a variety of detector devices. Exemplary detection methods include optical detection (e.g., fluorescence or chemiluminescence). As a non-limiting example, a fluorescent label can be detected using a detector device equipped with a module for generating excitation light that can be absorbed by the fluorophore, as well as a module for detecting the light emitted by the fluorophore.

[0082] In some embodiments, the detector further comprises the capability to handle partitioned samples (e.g., droplets), where individual partitioned samples enter the detector, undergo detection, and then exit the detector. In some embodiments, the partitioned samples (e.g., droplets) can be detected sequentially while the partitioned samples are flowing. In some embodiments, the partitioned samples (e.g., droplets) are arrayed on a surface, and the detector moves relative to the surface to detect a signal at each location containing a single partition. Examples of detectors are provided in International Publication No. 2010 / 036352, the contents of which are incorporated herein by reference. In some embodiments, detectable labels in the partitioned samples can be detected sequentially (e.g., using a chamber slide) without flowing the partitioned samples.

[0083] Following acquisition of the fluorescence detection data, a general-purpose computer system (referred to herein as a "host computer") can be used to store and process the data.

[0084] Also provided is the kit and mixture useful for carrying out the method.Kit can be, for example, comprised of a plastic container containing the mixture as described herein, optionally with instructions for its use.For example, in some embodiments, provide the mixture of forward primer or forward primer and reverse primer as described herein, which can be mixed by end user with RNaseH2 enzyme, probe, polymerase and / or other reagents as described herein.

[0085] In some embodiments, the mixture comprises at least a first forward primer comprising a 3' target-specific forward sequence and a reverse primer comprising a 3' target-specific reverse sequence, wherein the forward primer or reverse primer further comprises a first 5' universal sequence and a probe nucleic acid comprising: (i) a first fluorophore; (ii) a first quencher; (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the 5' universal sequence, or both; and (iv) at least one ribonucleotide separating the fluorophore and quencher. In some embodiments, the mixture comprises a first set of forward and reverse primers as described above, wherein each of the forward or reverse primers has the same 5' universal sequence but different (e.g., at least 2, 3, 4, 5, 8, 10, 15, 20, or more) 3' target-specific forward and 3' target-specific reverse sequences, respectively, such that at least 2, 3, 4, 5, 8, 10, 15, 20, or more different target nucleic acids can be amplified by the primers and detected by the probe nucleic acids.

[0086] In some embodiments, the mixture can contain two sets of primers: a first set (forward and reverse) of primers containing a first 5' universal sequence whose reverse complement is detectable by a first probe, and a second set (forward and reverse) of primers containing a second 5' universal sequence whose reverse complement is detectable by a second probe, along with at least two different, separately detectable probes. For each set of primers, either the forward primer or the reverse primer contains the 5' universal sequence, but not both. Either or both sets of primers can contain multiple different primers with different 3' target-specific sequences, allowing for amplification of different target nucleic acids within the same set of primers.

[0087] For example, in some embodiments, the mixture comprises: (a) a first set of forward primers comprising a 3' target-specific forward sequence and a reverse primer comprising a 3' target-specific reverse sequence, wherein the forward primers or reverse primers of the first set further comprise a first 5' universal sequence, and the first set targets a first plurality of different target nucleic acids; a first set of probe nucleic acids comprising: (i) a first fluorophore; (ii) a first quencher; (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of a first 5' universal sequence, or both; and (iv) at least one ribonucleotide separating the fluorophore and quencher; (b) a second set of forward primers comprising a 3' target-specific forward sequence and a reverse primer comprising a 3' target-specific reverse sequence, wherein the forward primers or reverse primers of the second set further comprise a second 5' universal sequence, and the second set targets a second plurality of different target nucleic acids; a second set of probe nucleic acids comprising (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of a second 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and quencher; This allows a first amplicon derived from a first set of forward and reverse primers and having a first 5' universal sequence to be distinguished from a second amplicon derived from a second set of forward and reverse primers and having a second 5' universal sequence based on the signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

[0088] The mixture can further include a third, fourth, fifth, sixth, or more sets of primers and probes, such as those described above, but targeting different target nucleic acids, with probes that detect different universal sequences as described herein, allowing for even higher degrees of multiplexing. [Example]

[0089] Figure 3 shows results from a single primer pair with a forward primer containing a 5' universal sequence tested on a Bio-Rad QX600 Droplet Digital PCR system using molecular beacons with one RNA base in some reaction wells and linear probes with an RNA base in other reaction wells. The test design evaluated different types of RNase H2 and also compared different types of RNase H2 against an RNA base probe with no addition of RNase H2 to the reaction.

[0090] The 20 μl reaction contained 5 μl of ddPCR supermix, 0.27 μl of DTT, 90 nM forward primer, 900 nM reverse primer, 500 nM fluorescent probe, 0.25 μl of RNase H2, 5 ng of genomic DNA, and nuclease-free water to adjust the final reaction volume to 20 μl. In reaction wells without RNase H2, 0.25 μl of nuclease-free water was added instead. In reaction wells with RNase H2, the following enzymes and concentrations were tested: NEB RNase H2 (catalog no. M0288S) at 50 mU per reaction and IDT RNase H2 (catalog no. 11-03-02-02) at 50 mU per reaction. Thermostable RNase H enzymes were also tested: NEB Thermostable RNase H (catalog no. M0523S) at 50 mU per reaction. Once the ddPCR reactions were set up, droplets were generated using a Bio-Rad AutoDG instrument and thermocycled on a Bio-Rad C1000 thermocycler using the following cycle: 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 59°C for 1 minute, a final denaturation step at 98°C for 10 minutes, and cooling to 4°C at a ramp rate of 2.5°C / second. Droplet fluorescence was measured using a Bio-Rad QX600 reader.

[0091] Tests were performed using HEX (Figure 3, Panel A) and FAM (Figure 3, Panel B) fluorophores. A greater than two-fold increase in fluorescence of positive droplets was measured for both molecular beacons and linear probes bearing RNA bases in the presence of IDT RNase H2 compared to reactions without RNase H2. Only limited or no fluorescence increase was observed with NEB RNase H2 and NEB thermostable RNase H.

[0092] Figure 4 displays the results from a 60-plex run on a Bio-Rad QX600 Droplet Digital PCR System, comparing standard and RNase H2-cleavable molecular beacons. The 60-plex run (shown in Figure 3) consists of 10 primer pairs to detect target 1 in the HEX channel, 10 primer pairs to detect target 2 in the FAM channel, 10 primer pairs to detect target 3 in the Cy5 channel, 10 primer pairs to detect target 4 in the Cy5.5 channel, 10 primer pairs to detect target 5 in the ROX channel, and 10 primer pairs to detect target 6 in the Atto590 channel. Within each set of 10 primer pairs, all forward primers have the same 5' universal sequence, so they are detected with the same molecular beacon probe. For example, if MB01 (see Table 1) is used to detect target 1, all 10 forward primers used for target 1 will have a 5' universal sequence (see Table 3) compatible with MB01. The same logic applies to each fluorescence channel. In this case, only six fluorescent probes are used for detection, despite having a total of 60 primer pairs in the ddPCR reaction across six fluorescent channels.

[0093] The ddPCR reactions contained 5 μl of ddPCR supermix, 0.27 μl of DTT, 90 nM of each forward primer, 900 nM of each reverse primer, 500 nM of each fluorescent probe, 100 mU of IDT RNase H2 (catalog no. 11-03-02-02), 5 ng of genomic DNA, and nuclease-free water to adjust the final reaction volume to 20 μl. In reaction wells with standard molecular beacons, RNase H2 was replaced with 0.25 μl of nuclease-free water. Once the ddPCR reactions were set up, droplets were generated using a Bio-Rad AutoDG instrument, and the droplets were thermocycled on a Bio-Rad C1000 thermocycler using the following cycles: 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 59°C for 1 minute, a final denaturation step at 98°C for 10 minutes, and cooling to 4°C at a ramp rate of 2.5°C / second. Droplet fluorescence was measured using a Bio-Rad QX600 reader. 1D histogram plots show droplet fluorescence amplitude on the X-axis versus droplet number on the Y-axis. Peaks labeled with stars correspond to negative droplets lacking target DNA. Peaks labeled with triangles correspond to positive droplets containing at least one copy of target DNA. The results show that the fluorescence amplitude separation between negative and positive droplet clusters increases more than two-fold with RNase H2-cleavable molecular beacons compared to standard molecular beacons. The diffusion of positive droplet clusters is also greater in this configuration. This demonstrates the increased fluorescence signal using molecular beacons with RNA bases combined with the use of RNase H2 in ddPCR reactions compared to standard molecular beacons for highly multiplexed applications.

[0094] A 96-plex with 16 primer pairs per channel across six channels and a 120-plex with 20 primer pairs per channel across six channels were tested using the same configuration as described for the 60-plex (data not shown).

[0095] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof and are to be included within the spirit and purpose of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for detecting a target nucleic acid in a sample, comprising: (a) forming a reaction mixture, said reaction mixture comprising: a sample nucleic acid; a plurality of forward primers comprising a 3' target-specific forward sequence; a plurality of reverse primers comprising a 3' target-specific reverse sequence, wherein the forward primer or the reverse primer further comprises a 5' universal sequence; a probe nucleic acid comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the 5′ universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; a DNA polymerase; RNase H2 enzyme and and (b) annealing the forward primer to a target nucleic acid in the sample nucleic acid and extending the forward primer with a polymerase using the target nucleic acid as a template to form a first strand extension product; (c) annealing the reverse primer to the first strand extension product and extending the reverse primer with the polymerase using the first strand extension product as a template to form a second strand extension product; when the reverse primer comprises the 5' universal sequence, the first strand extension product comprises the reverse complement of the 5' universal sequence, and when the forward primer comprises the 5' universal sequence, the second strand extension product comprises the reverse complement of the 5' universal sequence. That and; (d) annealing the probe nucleic acid to the reverse complement of the 5′ universal end sequence, and causing the RNase H2 enzyme to cleave the annealed probe at the ribonucleotide, thereby separating the quencher from the fluorophore and generating a detectable signal indicative of the presence of the target nucleic acid; A method comprising:

2. 2. The method of claim 1, wherein (a) through (d) occur within partitions, and the target nucleic acid is distributed among the partitions such that at least a portion of the partitions do not contain the target nucleic acid.

3. The method of claim 1 , wherein the sample nucleic acid is cell-free DNA.

4. 4. The method of claim 3, wherein the sample nucleic acid is from a pregnant woman and contains maternal and fetal DNA.

5. The method of claim 2 , wherein the partitions are droplets.

6. The method of claim 2, wherein the partition is a microwell.

7. 7. The method of claim 6, wherein the microwell is sealed by a lid, a cavity, or an oil layer.

8. The method of claim 1 , wherein the detectable signal is monitored in real time.

9. The method of claim 1 , wherein the detectable signal is monitored at an endpoint of the method.

10. The method of any one of claims 1 to 8, wherein the forward primer comprises the 5' universal sequence and the concentration of the reverse primer is higher than the concentration of the forward primer.

11. The method of any one of claims 1 to 8, wherein the reverse primer comprises the 5' universal sequence and the concentration of the forward primer is higher than the concentration of the forward primer.

12. The method of any one of claims 1 to 10, wherein the forward primer comprises the 5' universal sequence and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid.

13. 13. The method of any one of claims 1 to 8 or 12, wherein the reverse primer comprises the 5' universal sequence and the forward primer has a 5' tail sequence that does not anneal to the target nucleic acid.

14. The method according to any one of claims 1 to 12, wherein the probe nucleic acid is a linear probe.

15. The method of claim 14, wherein the linear probe comprises 80 to 100% of the 5' universal sequence.

16. 16. The method of claim 15, wherein the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, 30) nucleotides that anneal to the reverse complement of the 5' universal sequence.

17. 15. The method of claim 14, wherein the forward primer comprises the 5' universal sequence, and the linear probe and the reverse complement of the 5' universal sequence on the second strand extension product form a duplex that has a higher melting temperature than a duplex formed from the 5' universal sequence of the forward primer and the reverse complement of the 5' universal sequence.

18. 15. The method of claim 14, wherein the reverse primer comprises the 5' universal sequence, and the linear probe and the reverse complement of the 5' universal sequence on the first strand extension product form a duplex that has a higher melting temperature than a duplex formed from the 5' universal sequence of the reverse primer and the reverse complement of the 5' universal sequence.

19. 13. The method of any one of claims 1 to 12, wherein the probe nucleic acid forms a stem-loop and comprises, from 5' to 3', a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the ribonucleotide is in the loop sequence, and the 5' universal sequence comprises at least a portion of the loop sequence.

20. 20. The method of claim 19, wherein the 5' universal sequence further comprises at least a portion of the second stem sequence.

21. 20. The method of claim 19, wherein the first stem sequence and the second stem sequence are each 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides in length.

22. 22. The method of claim 19 or 21, wherein the loop sequence is 5 to 50 (e.g., 10 to 40 or 17 to 32) nucleotides in length.

23. The method of any one of claims 19 to 22, wherein the 5' universal sequence comprises all of the loop sequence, all of the second stem sequence, or all of the loop sequence and second stem sequence.

24. 24. The method of any one of claims 1 to 23, wherein the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers, allowing amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

25. 25. The method of any one of claims 1 to 24, wherein the RNase H2 enzyme is a Pyrococcus abyssi RNase H2 enzyme or a mutant thereof, a Pyrococcus furiosis RNase H2 enzyme or a mutant thereof, a Pyrococcus horikoshii RNase H2 enzyme or a mutant thereof, a Thermococcus kodakarensis RNase H2 enzyme or a mutant thereof, or a Thermococcus litoralis RNase H2 enzyme or a mutant thereof.

26. the reaction mixture (a) the first set of forward and reverse primers, the first set targets a first plurality of different target nucleic acids; and the forward or reverse primer of the first set comprises a first 5' universal sequence; and the first set of probe nucleic acids comprises (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the first 5' universal sequence, or both; a first set; (b) a second set of the forward and reverse primers, the second set targets a second plurality of different target nucleic acids; and the forward or reverse primer of the second set comprises a second 5' universal sequence that is different from the first 5' universal sequence; and the second set of probe nucleic acids comprises (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the second 5' universal sequence, or both; The second set and Including, This allows a first amplicon derived from the first set of forward and reverse primers and having the first 5' universal sequence to be distinguished from a second amplicon derived from the second set of forward and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

26. The method according to any one of claims 1 to 25.

27. The method of any one of claims 1 to 26, wherein the DNA polymerase lacks 5'-3' exonuclease activity.

28. A reaction mixture comprising: a plurality of forward primers comprising a 3' target-specific forward sequence; a plurality of reverse primers comprising a 3' target-specific reverse sequence, wherein the forward primer or the reverse primer further comprises a 5' universal sequence; a probe nucleic acid comprising (i) a fluorophore, (ii) a quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the 5′ universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; a DNA polymerase; RNase H2 enzyme and A reaction mixture comprising:

29. 30. The reaction mixture of claim 28, further comprising a sample nucleic acid.

30. 30. The reaction mixture of claim 29, wherein the sample nucleic acid is cell-free DNA.

31. 31. The reaction mixture of claim 30, wherein the sample nucleic acid is from a pregnant woman and contains maternal and fetal DNA.

32. 30. The reaction mixture of claim 28, wherein the reaction mixture is in partitions and the target nucleic acid is distributed among the partitions such that at least a portion of the partitions do not contain the target nucleic acid.

33. 33. The reaction mixture of claim 32, wherein the partitions are droplets.

34. 33. The reaction mixture of claim 32, wherein the partition is a microwell.

35. 35. The reaction mixture of claim 34, wherein the microwell is sealed by a lid, a cavity, or an oil layer.

36. 36. The reaction mixture of any of claims 28 to 35, wherein the forward primer comprises the 5' universal sequence and the concentration of the reverse primer is greater than the concentration of the forward primer.

37. 36. The reaction mixture of any of claims 28 to 35, wherein the reverse primer comprises the 5' universal sequence and the concentration of the forward primer is greater than the concentration of the forward primer.

38. 37. The reaction mixture of any one of claims 28 to 36, wherein the forward primer comprises the 5' universal sequence and the reverse primer has a 5' tail sequence that does not anneal to the target nucleic acid.

39. 38. The reaction mixture of any one of claims 28 to 35 or 37, wherein the reverse primer comprises the 5' universal sequence and the forward primer has a 5' tail sequence that does not anneal to the target nucleic acid.

40. The reaction mixture of any one of claims 28 to 39, wherein the probe nucleic acid is a linear probe.

41. 41. The reaction mixture of claim 40, wherein the linear probe comprises 80 to 100% of the 5' universal sequence.

42. 42. The reaction mixture of claim 41, wherein the probe nucleic acid has at least 10 (e.g., at least 15, 20, 25, 30) nucleotides that anneal to the reverse complement of the 5' universal sequence.

43. 40. The reaction mixture of any one of claims 28-39, wherein the probe nucleic acid forms a stem-loop and comprises, from 5' to 3', a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the ribonucleotide is in the loop sequence, and the 5' universal sequence comprises at least a portion of the loop sequence.

44. 44. The reaction mixture of claim 43, wherein the 5' universal sequence further comprises at least a portion of the second stem sequence.

45. 44. The reaction mixture of claim 43, wherein the first stem sequence and the second stem sequence are each 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides in length.

46. 46. ​​The reaction mixture of any one of claims 43 to 45, wherein the loop sequence is 5 to 50 (e.g., 10 to 40 or 17 to 32) nucleotides in length.

47. 46. ​​The reaction mixture of any one of claims 43-45, wherein the 5' universal sequence comprises all of the loop sequence, all of the second stem sequence, or all of the loop sequence and second stem sequence.

48. 48. The reaction mixture of any one of claims 28-47, wherein the plurality of forward primers comprises at least two (e.g., at least 3, 5, 10, 20, 30, 40, 50) different forward primers having five different 3' target-specific sequences, and the plurality of reverse primers comprises at least five different reverse primers, allowing amplification of two (e.g., at least 3, 5, 10, 20, 30, 40, 50) target nucleic acids.

49. 49. The reaction mixture of any one of claims 28 to 48, wherein the RNase H2 enzyme is a Pyrococcus abyssi RNase H2 enzyme or a variant thereof, a Pyrococcus furiosis RNase H2 enzyme or a variant thereof, a Pyrococcus horikoshii RNase H2 enzyme or a variant thereof, a Thermococcus kodakarensis RNase H2 enzyme or a variant thereof, or a Thermococcus litoralis RNase H2 enzyme or a variant thereof.

50. the reaction mixture (a) the first set of forward and reverse primers, the first set targets a first plurality of different target nucleic acids; and the forward or reverse primer of the first set comprises a first 5' universal sequence; and the first set of probe nucleic acids comprises (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the first 5' universal sequence, or both; a first set; (b) a second set of the forward and reverse primers, the second set targets a second plurality of different target nucleic acids; and the forward or reverse primer of the second set comprises a second 5' universal sequence that is different from the first 5' universal sequence; and the second set of probe nucleic acids comprises (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the second 5' universal sequence, or both; The second set and Including, This allows a first amplicon derived from the first set of forward primers and reverse primers and having the first 5' universal sequence to be distinguished from a second amplicon derived from the second set of forward primers and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively.

50. The reaction mixture of any one of claims 28 to 49.

51. 51. The reaction mixture of any one of claims 28 to 50, wherein the DNA polymerase lacks 5'-3' exonuclease activity.

52. A mixture comprising: (a) a first set of forward primers comprising a 3′ target-specific forward sequence and reverse primers comprising a 3′ target-specific reverse sequence, wherein the forward primers or the reverse primers of the first set further comprise a first 5′ universal sequence, and the first set targets a first plurality of different target nucleic acids; a first set of probe nucleic acids comprising (i) a first fluorophore, (ii) a first quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the first 5' universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; (b) a second set of forward primers comprising a 3′ target-specific forward sequence and reverse primers comprising a 3′ target-specific reverse sequence, wherein the forward primers or the reverse primers of the second set further comprise a second 5′ universal sequence, and the second set targets a second plurality of different target nucleic acids; a second set of probe nucleic acids comprising (i) a second fluorophore, (ii) a second quencher, (iii) at least 25% (e.g., at least 50%) or at least 10 (e.g., at least 15, 20, 25, or more) nucleotides of the second 5′ universal sequence, or both, and (iv) at least one ribonucleotide separating the fluorophore and the quencher; Including, This allows a first amplicon derived from the first set of forward primers and reverse primers and having the first 5' universal sequence to be distinguished from a second amplicon derived from the second set of forward primers and reverse primers and having the second 5' universal sequence based on signals from the first set of probe nucleic acids and the second set of probe nucleic acids, respectively. mixture.

53. 53. The mixture of claim 52, further comprising an RNase H2 enzyme.

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