Universal hairpin probes

Abstract

The disclosure relates to universal hairpin probes and methods of using the same. In some embodiments, the probes comprise a hybridization region that is readily altered to work with any desired target nucleic acid sequence, while leaving a secondary structure stem / loop region universal across different assays. The probes are thus advantageous for multiplexed detection of numerous targets.

Description

[0001]ALERE-40207.601 UNIVERSAL HAIRPIN PROBES CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application No. 63 / 551,384 filed February 8, 2024, and U.S. Provisional Patent Application No. 63 / 558,341 filed February 27, 2024, each of which is incorporated by reference herein in its entirety. FIELD Provided herein are universal hairpin probes and methods of using the same. The probes comprise a hybridization region that is readily altered to work with any desired target nucleic acid sequence, while leaving a secondary structure stem / loop region that is universal for different assays. BACKGROUND Nucleic acid analysis is a critical technique used in many fields, including biological research and clinical screening and diagnostics. Nucleic acid analysis can involve many different processes including amplification, hybridization, and sequencing. A variety of reagents may be employed in these methods, including nucleic acid probes, enzymes, buffers, salts, and detectable labels, such as fluorescent, colorometric, or luminescent labels. Significant efforts have been expended over many decades to optimize and improve nucleic acid analysis techniques to provide more accurate, more flexible, less expensive, higher throughput, more scalable, and more rapid technologies. While significant strides have been made, further improvements are still needed. SUMMARY Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids. Molecular beacons are generally hairpin- shaped molecules with an internally quenched fluorophore whose fluorescence is restored when the probe binds to a target nucleic acid sequence. Molecular beacons provide a non-radioactive method for detecting specific sequences of nucleic acids. They often find use in situations where ALERE-40207.601 it is either not possible or not desirable to isolate the probe-target hybrids from an excess of the hybridization probes. The primary drivers for selecting a nucleic acid amplification and / or detection technology often include: 1) performance (high sensitivity and specificity); 2) speed (often as low as 15 minutes or less or even 5 minutes or less are desired); 3) low cost; 4) scalability, including the ability to work with very small volumes and the ability to work in high throughput platforms such as chip-based detection formats; and 5) ability to multiplex (including spatial multiplexing). The probes provided herein, referred to as universal hairpin probes, accommodate these requirements by providing highly flexible probe design for use against multiple different target nucleic acid sequences, while providing a universal detection system that is particularly well suited for high throughput and multiplex detection systems. The use of such probes avoids the need to optimize the secondary structure of a molecular beacon for each different target detected. This optimization often presents the most difficult aspect of molecular beacon probe design and can be especially challenging for isothermal technologies. Definitions 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. In case of conflict, the present document, including definitions, will control. Preferred 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 present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entireties. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of”, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. ALERE-40207.601 For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. The terms, “fluorophore,” “fluorescent moiety,” “fluorescent label,” and “fluorescent dye” are used interchangeably herein and refer to a molecule that absorbs a quantum of electromagnetic radiation at one wavelength, and emits one or more photons at a different, typically longer, wavelength in response thereto. The terms “quencher”, “quencher dye”, and “quencher moiety” are used interchangeably herein and refer to a moiety that absorbs excitation energy from a fluorophore. In some embodiments, a probe described herein comprises both a “fluorophore” and a “quencher”. Binding of a target nucleic acid to the probe influences the distance and / or interaction between the fluorophore and the quencher, thereby modulating the detectable fluorescent signal in a sample. As used herein, “hybridization” and “binding” are used interchangeably and refer to the non-covalent binding or “base pairing” of complementary nucleic acid sequences to one another. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. The term “complementary” as used herein refers to substantial complementarity (e.g. two strands containing at least 50% complementary bases (e.g. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% complementary bases) or full complementarity (e.g. complete complementarity, or two strands that are 100% complementary to one another). Whether or not a particular probe remains base paired with a polynucleotide sequence depends on the degree of complementarity, the length of the probe, and the stringency of the binding conditions. The higher the stringency, the higher must be the degree of complementarity, and / or the longer the probe for binding or base pairing to remain stable. As used herein, “stringency” refers to the combination of conditions to which nucleic acids are subjected that cause double-stranded nucleic acid to dissociate into component single strands such as pH extremes, high temperature, and salt concentration. The phrase “high stringency” refers to hybridization conditions that are sufficiently stringent or restrictive such that only specific base pairings will occur. The specificity should be sufficient to allow for the ALERE-40207.601 detection of unique sequences using an oligonucleotide probe or closely related sequence under standard Southern hybridization protocols (as described in J. Mol. Biol. 98:503 (1975)). The term “nicking” refers to the cleavage of only one strand of the double-stranded portion of a fully or partially double-stranded nucleic acid. A “nicking enzyme” refers to a protein, that in its native form, is capable of binding to a fully or partially double-stranded nucleic acid (e.g. DNA) and cleaving one strand of the double-stranded duplex. The position where the nucleic acid is nicked is referred to as the nicking site or nicking enzyme site. The recognition sequence that the nicking enzyme recognizes is referred to as the nicking enzyme binding site. As used herein, the term nicking enzyme includes modified (i.e., non-native) nicking enzymes that have reduced or no nicking activity (e.g., having one more sequence variations that reduce or eliminate nicking activity). The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including modifications thereof. These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, for example, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like). In some embodiments, “nucleic acid” refers to modified DNA or modified RNA. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5- iodo-uracil, backbone modifications, methylations, and unusual base-pairing combinations, such as the isobases. The ”nucleic acids” described herein include not only the standard bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) but also non-standard or non-natural nucleotides. Non-standard or non-natural nucleotides, which form hydrogen-bonding base pairs, ALERE-40207.601 are described, for example, in U.S. Pat. Nos. 5,432,272, 5,965,364, 6,001,983, 6,037,120, and 6,140,496, all of which are incorporated herein by reference. By “non-standard nucleotide” or “non-natural nucleotide” it is meant a base other than A, G, C, T, or U that is susceptible to incorporation into an oligonucleotide and that is capable of base-pairing by hydrogen bonding, or by hydrophobic, entropic, or van der Waals interactions, with a complementary non-standard or non-natural nucleotide to form a base pair. Some examples include the base pair combinations of iso-C / iso-G, K / X, K / P, H / J, and M / N, as illustrated in U.S. Pat. No. 6,037,120, incorporated herein by reference. Other non-natural nucleotides for use in oligonucleotides include, for example, naphthalene, phenanthrene, and pyrene derivatives as discussed, for example, in Ren, et al., 1996 and McMinn et al., 1999, both of which are incorporated herein by reference. These bases do not utilize hydrogen bonding for stabilization, but instead rely on hydrophobic or van der Waals interactions to form base pairs. The term “oligonucleotide,” as used herein, refers to a short nucleic acid sequence comprising from about 2 to about to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values). The term “sample” is used in the broadest sense and is inclusive of any sample type that contains or is suspected of containing a target nucleic acid sequence. In some embodiments, the sample is a biological sample. For example, in some embodiments the sample may be a biological sample such as blood, serum, buffy coat, plasma, tissue, cerebrospinal fluid, interstitial fluid, saliva, placental tissue, meconium, cord blood, bone marrow, breast milk, bronchoalveolar lavage, cell lines, exhaled air, feces, ascites, pleural fluid, synovial fluid, hair, nail clippings, semen, or urine. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows that a UHP designed for detection of COVID compares to the performance of the gold-standard molecular probe in a proof-of-concept study. (FIG. 1A) COVID UHP scheme with COVID amplicon product (top) and UHP design (bottom, right) compared to the gold-standard MB design (bottom, left). (FIG. 1B) MB performance compared with UHP on a ALERE-40207.601 qPCR fluorescent reader. All curves assert at a similar time. (FIG. 1C) IDNOW test (n=20) of UHP (blue) vs MB (black) in NP matrix. SARS-CoV-2 virus was added at 0.5X LOD, and as such, not all replicates were expected to be detected. e. NEAR reactions containing the UHP recorded 30% more positive calls than NEAR reactions containing the standard MB (15 vs 8 assertions). [MB: Molecular Beacon; UHP: Universal Hairpin Probe; NTC: Non-Target Control;; Handle: Hairpin; T1 / T2: Template 1 / 2; BHQ1: Black Hole Quencher 1; FAM: Fluorescein; NP: nasopharyngeal] FIG. 2 UHPs obviate the need to balance MB stem vs target hybridization energies. Instead, a universal, optimized stem design can be used for almost any DNA target of interest, making the design both rapid and simple. Four additional assays comparing UHP to MB performance (n=3) are shown. All four UHPs were rapidly designed and showed comparable performance to MBs without the need to perform further design optimizations. [TTA: Time to Assertion; Time To Assertion is a metric analogous to a Ct (Cycle Threshold) in qPCR. Because there are no true “cycles” in isothermal amplification technologies, the Time to Assertion marks the time at which exponential amplification begins. StrepA is a 6 minute assay while FluA, RSVA and RSVB are 10 minute assays.] FIG. 3 shows that UHPs have a unique mechanism of action (IC: Internal Control Target; NE: Nicking Enzyme; pol: Polymerase)). (FIG. 3A) UHPs require polymerase to open. In the presence of high quantities of amplification target, without polymerase, MB opens (black). UHPs on the other hand, remain closed (blue). (FIG. 3B) UHPs require NE to open. Two models were constructed to demonstrate the dependance of UHPs on NE. (i.) Model “End” displays a UHP pre-hybridized to a simulated target with the 3’ end of the target fully complementary to the UHP spacer region. Model “Bothsides” displays a UHP pre-hybridized to a simulated target with the 3’ end of the target extending beyond the UHP spacer region and complementary to any region within the UHP. ii) Both models are readily opened in the presence of NE, even without the presence of templates because the target is prehybridized to the UHP. Conversely, when all components of the NEAR reaction are present except NE, both models remain closed. In scheme, Blue: Hairpin; Black: model hybriziation sequence; Orange: spacer region. (iii.) Two dsDNA cutting restriction enzymes with the same cut site currently employed in NEAR, did not open the UHP. In contrast another NE, NE-B,, which recognizes a a different nicking recognition site, did ALERE-40207.601 open the model UHPs, albeit later, despite that nicking recognition site sequence being absent in the model systems. (FIG. 3C) (i) Scheme for transitional hybridization between NEAR product and UHP in the reaction. (ii) Proposed final product for open UHP facilitated by NE and DNA pol. (FIG. 3D) Mass spectrometry confirmation of the presence of UHP product (blue), shown in Figure 3Cii, in a completed COVID reaction containing NE. The open and extended UHP product is missing from the negative control without NE, further supporting the proposed mechanism. FIG. 4 shows that the transition of UHP from ssDNA to dsDNA on opening has unique benefits for detection. (FIG. 4A) Left: Fluorescent curves for target detection. Right: Melting temperature curves for +target curves. Despite having the same spacer region, open UHP has a much higher Tm, indicating the extended product formed during UHP opening has more base pairs than the open MB. This observation provides further evidence for the proposed mechanism of opening and extension of the UHP observed in Fig. 3D. (FIG. 4B) The extention of the UHP product helps in recognition of either the forward of reverse product (P1 or P2) regardless of product concentration. (Left) For a traditional MB designed to detect P1 (black), P1 dominates the early reaction in a 4F:1R template skewed reaction, with P2 catching up at ~7min and competing P1 off the MB, indicated by the drop in MB signal. By contrast, UHPs designed to detect P1 (Blue) or P2 (Red) are extended when opened making the region of complimentary longer, greatly reducing competition from amplification products alone (Right) In the opposite template skew 1F:4R,both UHPs clearly show the presence of product at two minutes (blue), but, the MB remains closed for for 12min. DETAILED DESCRIPTION In some aspects, provided herein are nucleic acid probes. In some embodiments, provided herein are nucleic acid probes possessing a stem-loop structure (i.e. a hairpin structure). The nucleic acid probes described herein are also referred to as “universal hairpin probes”, or “UHPs”. Hairpin probes are hair-pin shaped oligonucleotides containing a fluorophore and a quencher. Molecular beacon probes are an example of a type of hairpin probe known in the field. In many existing molecular beacon probes, the loop of the hair-pin contains a probe sequence that is complementary to a target sequence and the stem is formed by annealing of ALERE-40207.601 complementary arm sequences located on either side of the probe sequence. A fluorophore and a quenching molecule are covalently linked at opposite ends of each arm. Under conditions that prevent the oligonucleotides from hybridizing to its complementary target or when the molecular beacon is free in solution, the fluorescent and quenching molecules are proximal to one another, thereby preventing fluorescence resonance energy transfer (FRET). When the molecular beacon encounters a target molecule with sequence homology to the molecular beacon, hybridization occurs, and the loop structure is converted to a stable more rigid conformation causing separation of the fluorophore and quencher molecules leading to fluorescence (Tyagi et al. Nature Biotechnology 14: March 1996, 303-308, herein incorporated by reference in its entirety). When used to detect target nucleic acid molecules during an amplification reaction, due to the specificity of the probe, the generation of fluorescence occurs upon hybridization of the intended amplified product and the probe. Molecular beacons are extraordinarily specific and can discern a single nucleotide polymorphism. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, enabling several products in the same reaction to be quantitated simultaneously. For quantitative amplification processes, molecular beacons can specifically bind to the amplified target following each cycle of amplification, and because non-hybridized molecular beacons are dark, it is not necessary to isolate the probe-target hybrids to quantitatively determine the amount of amplified product. The resulting signal is proportional to the amount of target or amplified product. This can be done in real time. As with other real time formats, the specific reaction conditions should be optimized for each primer / probe set to ensure accuracy and precision. However, typical molecular beacon probes require design of a loop structure that binds to the target molecule while also taking into consideration the secondary structure and thermodynamically favored interactions, which is challenging and time consuming. Moreover, the melting temperatures of the stem and loop sequences should be highly correlated to the assay temperature and are affected by the use of various fluorophores and quenchers, further complicating molecular beacon design. Accordingly, multiplexed assays using molecular beacon probes require a delicate balance and iterative testing during the design process and can be difficult to optimize. . In contrast, for the universal hairpin probes described herein, the target binding sequence is not present in the loop structure of the probe, thus simplifying the design process and more readily enabling multiplexed designs. ALERE-40207.601 In some aspects, provided herein is nucleic acid probe comprising a first region having a 3’ end having a sequence complementary to at least a portion of a target nucleic acid sequence. The nucleic acid probe further comprises a second region 5’ of the first region having a first hairpin forming sequence. The nucleic acid probe further comprises a third region 5’ of the second region having a loop structure. The nucleic acid probe further comprises a fourth region 5’ of the third region having a second hairpin forming sequence. The fourth region additionally comprises a 5’ end. In some embodiments, the second hairpin forming sequence, under reactions conditions, hybridizes to the first hairpin forming sequence. Accordingly, in the absence of a target nucleic acid molecule, the first and second hairpin forming sequences hybridize to form the “stem” portion of the hairpin probe (e.g. the “stem” portion of the “stem-loop” structure). The second hairpin forming sequence does not hybridize to the first region. Accordingly, the first region overhangs from the stem portion of the hairpin and is available to bind to the target nucleic acid, when present in the sample. In some embodiments, the probes comprise a label. For example, in some embodiments, the probes comprise a fluorescent label. In some embodiments, the probes comprise a capture moiety to facilitate isolation of the probe, and any molecules bound thereto or generated therefrom. In some embodiments, where the probe comprises a fluorescent label, it further comprises a quencher. In some embodiments, the fluorophore is attached to the second region. In some embodiments, the fluorophore is attached to the fourth region. The quencher moiety is attached to the other of the second of the fourth region. Accordingly, in some embodiments the fluorophore is attached to the second region and the quencher is attached to the fourth region. In other embodiments, the fluorophore is attached to the fourth region and the quencher is attached to the second region. Any suitable fluorophore may be used in the nucleic acid probes described herein. For example, the fluorophore may be a fluorescein-family dye, polyhalofluorescein-family dye, hexachlorofluorescein-family dye, coumarin-family dye, rhodamine-family dye, cyanine-family dye, oxazine-family dye, thiazin-family dye, squaraine-family dye, chelated lanthanide-family dye, azo-family dye, triphenylmethane-family dye, or a BODIPYO-family dye. Examples of ALERE-40207.601 fluorophores include, but are not limited to, FAME, HEXTm, JOETm, NEDTm, PET, ROXTM, TAMRATm, TETTm, TEXAS RED, and VIC. Any suitable quencher may be used in the nucleic acid probes described herein. The quencher may be selected from any suitable quencher known in the art, such as, for example, BLACK HOLE QUENCHER 1 (BHQ-19), BLACK HOLE QUENCHER 2 (BHQ-28), IOWA BLACK FQ, IOWA BLACK RQ,. ECLIPSE QUENCHER, AND MGB ECLIPSE QUENCHER. For example, an oligonucleotide probe may comprise a FAM fluorophore and a BHQ-quencher (e.g., BHQ-10, BHQ-28). In some embodiments, in the absence of a target nucleic acid sequence, the quencher moiety is proximal to the fluorophore, thereby preventing a detectable fluorescent signal. In some embodiments, when a target nucleic acid sequence binds to the first region of the nucleic acid probe, the nucleic acid probe undergoes a conformational change such that the quencher moiety is distanced from the fluorophore, thereby allowing a detectable fluorescent signal to be produced. The ability of the fluorophore to produce a measurable signal is dependent on the Förster resonance energy transfer between the fluorophore and the quencher. The efficiency of energy transfer is inversely proportional to the distance between the energy emitting and energy accepting molecules to the sixth power and is quantitatively described by the equation: See, e.g., Clegg R (2009) “Förster resonance energy transfer—FRET: what is it, why do it, and how it's done”, in Gadella TW (ed.) FRET and FLIM Techniques. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 33. Elsevier. pp. 1–57, incorporated herein by reference. In some embodiments, in the absence of a target nucleic acid sequence, the quencher moiety is distanced from the fluorophore, thereby allowing a detectable fluorescent signal to be produced. In some embodiments, when a target nucleic acid sequence binds to the first region of the nucleic acid probe, the nucleic acid probe undergoes a conformational change such that the ALERE-40207.601 quencher moiety is proximal to the fluorophore, thereby preventing a detectable fluorescent signal. The nucleic acid probes described herein can contain combinations of deoxyribo- and ribonucleotides. The nucleic acid probes described herein can contain any suitable combination of bases, including natural and non-natural nucleotides such as uracil, adenine, thymine, cytosine, guanine, inosine, isocytosine and isoguanine. In some embodiments, the nucleic acid probes described herein are oligonucleotide probes (e.g. probes comprising 2 to about 100 nucleotides). In some embodiments, the nucleic acid probes described herein comprise about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, about 75 nucleotides, about 80 nucleotides, about 85 nucleotides, about 90 nucleotides, about 95 nucleotides, or about 100 nucleotides. In some embodiments, the nucleic acid probes described herein comprise about 20 to about 60 nucleotides. For example, in some embodiments the nucleic acid probes described herein comprise 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. In some embodiments, the nucleic acid probe comprises 44 nucleotides. In some embodiments, the nucleic acid probes described herein contain more than 100 nucleotides. The first region may comprise any suitable number and combination of nucleotides, depending on the intended target sequence. The sequence of the first region varies depending on the target sequence to be detected. In some embodiments, the first region comprises 5-50 nucleotides. In some embodiments, the first region comprises 8-30 nucleotides. For example, in some embodiments the first region comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, or 30 nucleotides. In some embodiments, the first region comprises 11, 12, 13, 14, 15, or 16 nucleotides. In some embodiments, at least a portion of the target nucleic acid binds to the first region with substantial (e.g. more than 50%) complementarity. In some embodiments, a portion of the target nucleic acid binds to the first region with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 100% complementarity. In some embodiments, a portion of the target nucleic acid binds to the first ALERE-40207.601 region and a portion of the target nucleic acid binds to another region within the nucleic acid probe, such as the second region or the fourth region. In some embodiments, the portion of the target nucleic acid that binds to the nucleic acid probe is about 10 to about 50 nucleotides in length. In some embodiments, the portion of the target nucleic acid that binds to the nucleic acid probe is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 nucleotides in length. In some embodiments, the target nucleic acid comprises additional flanking sequences that do not bind to the nucleic acid probe. The second region and the fourth region may comprise any suitable number and combination of nucleotides and are optimized to facilitate hybridization between the first and second hairpin forming sequences in the absence of the target nucleic acid. In some embodiments, the sequence of the second region and the sequence of the fourth region are consistent (i.e. does not change) across multiple nucleic acid probes comprising multiple different first region sequences. In other words, in some embodiments the sequence of the second region (e.g. the first hairpin forming sequence) and the sequence of the fourth region (e.g. the second hairpin forming sequence) are consistent across multiple nucleic acid probes, even when the probes are designed to bind to different target analytes. In some embodiments, the second region and the fourth region each comprise 5-50 nucleotides. In some embodiments, the second region and the fourth region comprise the same number of nucleotides, such that perfect hybridization occurs between the first and second hairpin forming sequences. In some embodiments, the second comprises 5-25 nucleotides. In some embodiments, the fourth region comprises 5-25 nucleotides. In some embodiments, the second region and the fourth region each comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the second region and the fourth region each comprise 9 nucleotides. The first and second hairpin forming sequences may be designed such that the temperature required to separate the first and second hairpin forming sequences (e.g. the melting temperature (Tm) of the hybridized stem structure when the nucleic acid probe is not bound to the target sequence) is about 45°C to about 70°C. For example, the melting temperature of the hybridized stem structure may be about 45°C, about 50°C, about 55°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C. In some embodiments, the second region and the fourth region are ALERE-40207.601 optimized such that disruption of the stem structure upon addition of the target nucleic acid is thermodynamically favorable. The energy required to break the secondary structure is referred to as ΔG. A higher negative ΔG value in reference to the first and second hairpin forming sequences indicates a propensity for sequences to hybridize to each other, rather than to the target nucleic acid. Accordingly, a higher ΔG indicates a propensity for the stem structure to remain intact (e.g. for the first and second hairpin forming sequences to remain hybridized to one another), whereas a lower ΔG indicates a propensity for the hybridization between the first and second hairpin forming sequences to be disrupted upon addition of the target nucleic acid. The ΔG should be optimized such that the hairpin structure is stable in isolation (e.g. in the absence of the target nucleic acid), thus retaining the fluorophore and the quencher remain in proximity to one another in the absence of the target nucleic acid. Additionally, the ΔG should be optimized such that the hairpin structure thermodynamically unfavorable in the presence of the target nucleic acid, such that the target can bind to the first region and disrupt the hairpin structure, freeing the fluorophore from the quencher. In some embodiments, the second region and the fourth region are designed such that the ΔG (e.g. the energy required to break the stem structure after hybridization of the first and second hairpin forming sequences) is between about –5 kcal mol–1and about –20 kcal mol–1. In some embodiments, the ΔG is about –5 kcal mol–1, about –6 kcal mol–1, about –7 kcal mol–1, about –8 kcal mol–1, about –9 kcal mol–1, or about –10 kcal mol–1. The third region comprises a loop structure, which may contain any suitable number and composition of nucleotides. In some embodiments, the loop structure comprises 3-50 nucleotides. In some embodiments, the loop structure comprises 5-25 nucleotides (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). In some embodiments, the loop structure comprises 10-20 nucleotides. In some embodiments, the loop structure comprises 12 nucleotides. In some embodiments, the loop structure comprises more than 50 nucleotides. In contrast to standard molecular beacon probes, in which the loop structure is designed to be complementary to a target nucleic acid sequence, in some embodiments the loop structure in the probes described herein remains consistent across multiple probe types optimized for different target sequences. ALERE-40207.601 The nucleic acid probes described herein bind to a target nucleic acid sequence, if present in a sample. In some embodiments, in a reaction mixture comprising a nucleic acid probe described herein, a DNA polymerase, a nicking enzyme, and the target sequence, the first region of the nucleic acid probe binds to the target nucleic acid sequence and the probe undergoes a conformational shift from a “closed” ssDNA UHP to an “open” dsDNA UHP. This shift is also referred to herein as the probe “opening”. In some embodiments where fluorescent labels and quenchers are employed, in such a reaction mixture the first region binds to the target nucleic acid sequence and the probe undergoes a conformational shift from a “closed” ssDNA wherein the fluorophore is proximal to the quencher and no detectable fluorescent signal is observed, to an “open” dsDNA wherein the fluorophore is distanced from the quencher and a fluorescent signal can be detected. As shown in Example 1, a unique aspect of the UHPs described herein is that the nicking enzyme facilitates opening of the probe, even when no nicking site is present in the probe. While the invention is not limited to any specific mechanism of action and an understanding of the mechanism of action is not required to practice the invention, it is contemplated that the nicking enzyme, even lacking a nickable substrate, facilitates conformational changes in the nucleic acid that results in a successful UHP reaction. As such, the nicking enzyme provides a reaction facilitator. It is contemplated that other reaction facilitators may be employed, including modified nicking enzymes that have reduced or no nicking activity. In some embodiments, when the target nucleic acid sequence binds to the first region of the nucleic acid probe, the nucleic acid probe undergoes a conformational change such that the position and size of the loop structure is modified, thereby producing a second loop structure that is smaller than the loop structure originally present within the third region of the nucleic acid probe. For example, in some embodiments, the first loop structure comprises 10 or more nucleotides and the second loop structure comprises less than 10 nucleotides. For example, in some embodiments the loop structure comprises 12 nucleotides and the second loop structure comprises 5 nucleotides. In some embodiments, upon binding of the target nucleic acid sequence to the first region of the nucleic acid probe, the nucleic acid probe undergoes a conformational change such that a second, smaller hairpin structure is formed comprising both a smaller stem and a smaller loop compared to the hairpin structure originally present in the nucleic acid probe in the absence of ALERE-40207.601 the target nucleic acid. In some embodiments, a portion of the target nucleic acid binds to the first region and a portion of the target nucleic acid sequence is at least partially complementary to one of the hairpin forming sequences present in either the second region or the fourth region. Accordingly, in some embodiments binding of the target nucleic acid sequence to the first region of the nucleic acid probe disrupts the hybridization of the first and second hairpin forming sequences. In some embodiments, a portion of the target nucleic acid binds to the first region and a portion of the target nucleic acid binds to the second region, thus disrupting hybridization between the second region and the fourth region (e.g. the first hairpin forming sequence and the second hairpin forming sequence). In some embodiments, a portion of the target nucleic acid binds to the first region and a portion of the target nucleic acid binds to the fourth region, thus disrupting hybridization between the second region and the fourth region (e.g. the first hairpin forming sequence and the second hairpin forming sequence). In some embodiments, disruption of the binding between the first and second hairpin forming sequences causes a transformation in the size and location of the stem and / or loop of the hairpin structure. In some embodiments, disruption of the binding between the first and second hairpin forming sequences induces a shift in the loop structure, such that a portion of the loop structure is elongated and the location of the loop shifts within the nucleic acid probe. In some embodiments, a second loop structure is formed, replacing the original loop structure. In some embodiments, the second loop structure is smaller and comprises nucleotides that were not originally present within the loop structure in the absence of the target nucleic acid. For example, in some embodiments a second loop structure is formed, comprising nucleotides that were originally present in either the second or the fourth region, whichever does not bind to the target nucleic acid. For example, in some embodiments the target nucleic acid binds to the first region and the second region, inducing a transformation that creates a second, smaller loop comprising nucleotides that were originally present within the fourth region. As another example, in some embodiments the target nucleic acid binds to the first region and the fourth region, inducing a transformation that creates a second, smaller loop comprising nucleotides that were originally present within the second region. In some embodiments, disruption of the binding between the first and second hairpin forming sequences causes a transformation in the size and location of the stem structure. In some ALERE-40207.601 embodiments, the target nucleic acid binds to the first region and the second region, inducing a transformation such that a portion of the loop structure is elongated, forming a linear strand of unbound nucleotides. In some embodiments, disruption of the binding between the first and second hairpin forming sequences causes a transformation such that a portion of the second or the fourth region originally present within the nucleic acid probe in the absence of the target nucleic acid forms one strand of a second, smaller stem structure that binds to a complementary strand that was originally present within the loop structure of the nucleic acid probe in the absence of the target nucleic acid. In some embodiments, where fluorescent labels and quenchers are employed, the transformations described above induce distancing of the quencher and the fluorophore, thereby permitting a detectable fluorescent signal. In some embodiments, the transformations described above require the presence of a polymerase and a nicking enzyme, in addition to the target nucleic acid sequence, to facilitate the transformation. Suitable nicking enzymes and polymerases are described herein. In some embodiments, the nucleic acid probe is designed such that being bound to the target nucleic acid is more thermodynamically favorable than existing in the unbound state. For example, in some embodiments a higher temperature is required to disrupt the binding of the target nucleic acid to the nucleic acid probe (e.g. to the first region, or to the first region and the second region, or to the first region and the fourth region). For example, the temperature required to disrupt hybridization between the first and second hairpin forming sequences may be about 45°C to about 65°C, whereas the temperature required to disrupt hybridization between the target nucleic acid and the nucleic acid probe may be higher than 65°C. For example, the temperature required to disrupt hybridization between the target nucleic acid and the nucleic acid probe may be about 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C, 68.5°C, 69°C, 69.5°C, or about 70°C. In some embodiments, the energy required (e.g. the ΔG) to disrupt the secondary structure formed after binding of the target nucleic acid to the nucleic acid probe may be greater than the energy required to disrupt the hybridization between the first and second hairpin forming sequences. For example, the ΔG to disrupt the binding between the target nucleic acid and the nucleic acid probe may be greater than –10 kcal mol–1, greater than –15 kcal mol–1, greater than –20 kcal mol–1, greater than –25 kcal mol–1, greater than –26 kcal mol–1, greater than ALERE-40207.601 –27 kcal mol–1, greater than –28 kcal mol–1, greater than –29 kcal mol–1, greater than –30 kcal mol–1, greater than –31 kcal mol–1, greater than –32 kcal mol–1, greater than –33 kcal mol–1, greater than –34 kcal mol–1, greater than –35 kcal mol–1, greater than –36 kcal mol–1, greater than –37 kcal mol–1, greater than –38 kcal mol–1, greater than –39 kcal mol–1, or greater than –40 kcal mol–1. The nucleic acid probes described herein find use in various methods. For example, the nucleic acid probes described herein are useful for various methods involving amplification and / or detection of nucleic acid, including DNA or RNA, in a sample. The sample may be a biological sample (e.g. a bodily fluid, a tissue, etc.) obtained from a subject. “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject is a human. In some embodiments, the methods include amplifying nucleic acid. In some embodiments, the methods include detecting a target nucleic acid, if present in the sample, by measuring fluorescence in the sample. In some embodiments, the fluorescence in the sample is measured using a suitable instrument, such as a plate reader. In some embodiments, fluorescence in the sample is compared to a baseline or control level, such as a level of fluorescence in a negative control sample (e.g. a sample not containing the target nucleic acid). In some embodiments, an increased fluorescent signal indicates that the target nucleic acid is present in the sample, and that binding of the target nucleic acid has distanced the fluorophore and the quencher from one another. In some embodiments, a decreased fluorescent signal indicates that the target nucleic acid is present in the sample, and that binding of the target nucleic acid has brought the fluorophore and the quencher into proximity to one another. Exemplary methods include, for example, polymerase chain reaction-based methods (e.g. PCR, RT-PCR, hot-start PCR, long-range PCR, qPCR, RT-qPCR, quantitative PCR, digital PCR, nested PCR, hemi-nested PCR, semi-nested PCR, etc.), and isothermal amplification methods (e.g. nucleic acid sequence based amplification, loop-mediated isothermal amplification, strand displacement amplification, transcription-mediated amplification, single primer isothermal amplification, recombinase polymerase amplification, rolling circle ALERE-40207.601 amplification, etc.). In some embodiments, the nucleic acid probes described herein find use in nicking and extension reactions (NEAR), including those described in PCT Publication No. WO2009012246A2, U.S. Patent No. 11,186,864, U.S. Patent No. 10,329,601, and PCT Publication No. WO2019046610A1, the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the nucleic acid probes find use in genetic analysis, such as for gene copy determination, allele discrimination, SNP detection, genotyping, etc. In some embodiments, the nucleic acid probes find use in methods of detecting genetic variations in a sequence including substitutions, insertions, deletions, and the like. In some embodiments, the nucleic acid probes find use in genetic analysis of SNPs in a sample obtained from a subject. In some embodiments, the nucleic acid probes described herein may be used for detection and / or quantification of one or more pathogens in a sample. Any pathogen type may be detected and / or quantified, including algae, bacteria, fungi, prions, viroids, viruses, and other parasitic pathogens. For example, in some embodiments the nucleic acid probes described herein may be used for viral load quantification. In some embodiments, the nucleic acid probe is designed for detection of any pathogen in a sample by designing the sequence of the first region to bind to a portion of a target sequence of the pathogen. In some embodiments, the nucleic acid probe is designed for detection of a pathogen in a sample by designing the sequence of the first region and the second region to bind to a target sequence of the pathogen. In some embodiments, the nucleic acid probe is designed for detection of a pathogen in a sample by designing the sequence of the first region and the fourth region to bind to a target sequence of the pathogen. In some embodiments, the nucleic acid probe is designed for detection of an upper respiratory virus. In some embodiments, the nucleic acid probe is used for detection of an upper respiratory virus, such as influenza, respiratory syncytial virus, adenovirus, coxsackievirus, parainfluenza, metapneumovirus, enterovirus, coronavirus, or streptococcus. In some embodiments, a plurality of nucleic acid probes are used for multiplexed detection of a plurality of distinct upper respiratory viruses. In some embodiments, the nucleic acid probes described herein may be used in multiplexed methods. For example, the nucleic acid probes may be used for multiplexed detection of multiple pathogens. As another example, the nucleic acid probes may be used for ALERE-40207.601 multiplexed detection of multiple genetic variants (e.g. multiple SNPs). The nucleic acid probes described herein facilitate multiplexed detection by avoiding the need to design multiple third regions (e.g. loop structures) for different targets. In some aspects, provided herein is a reaction mixture comprising a nucleic acid probe as described herein (i.e. a universal hairpin probe), a polymerase, and a nicking enzyme. The polymerase may be any protein able to catalyze the specific incorporation of nucleotides to extend a 3′ hydroxyl terminus of a primer molecule against a target nucleic acid sequence. In some embodiments, the polymerase is thermophilic such that it is active at an elevated reaction temperature. The polymerase may have reverse transcription capabilities, such that the reaction mixture can amplify RNA targets without the use of a separate reverse transcriptase. In some embodiments, more than one polymerase is included in the reaction mixture. The polymerase may be any suitable polymerase, including but not limited to: Bst DNA polymerase, Bst DNA polymerase (Large fragment), 9°Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, Large (Klenow) fragment, Klenow fragment (3′-5′ exo–), T4 DNA polymerase, T7 DNA polymerase, Deep VentR ™ (exo–) DNA Polymerase, Deep VentR ™ DNA Polymerase, DyNAzyme ™ EXT DNA, DyNAzyme ™ II Hot Start DNA Polymerase, Phusion ™ High-Fidelity DNA Polymerase, Therminator ™ DNA Polymerase, Therminator ™ II DNA Polymerase, VentR ® DNA Polymerase, VentR ® (exo-) DNA Polymerase, RepliPHI ™ Phi29 DNA Polymerase, rBst DNA Polymerase, rBst DNA Polymerase, Large Fragment (IsoTherm ™ DNA Polymerase), MasterAmp ™ AmpliTherm ™ DNA Polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase Beta, and ThermoPhi DNA polymerase. The nicking enzyme may be any suitable nicking enzyme, including but not limited to: Nb.BbvCI, Nb.Bpu10I, Nb.BsaI, Nb.BsmI, Nb.BsrDI, Nb.BstNBIP, Nb.BstSEIP, Nb.BtsI, Nb.SapI, Nt.AlwI, Nt.BbvCI, Nt.BhaIIIP, Nt.Bpu10I, Nt.Bpu10IB, Nt.BsaI, Nt.BsmAI, Nt.BsmBI, Nt.BspD6I, Nt.BspQI, Nt.Bst9I, Nt.BstNBI, Nt.BstSEI, Nt.CviARORFMP, Nt.CviFRORFAP, Nt.CviPII, Nt.CviPIIm, Nt.CviQII, Nt.CviQXI, Nt.EsaSS1198P, Nt.MlyI, and Nt.SapI. In some embodiments, the reaction mixture additionally comprises a target nucleic acid sequence. For example, the reaction mixture may additionally comprise a target nucleic acid ALERE-40207.601 sequence from a pathogen of interest to be detected in the reaction mixture. Suitable target nucleic acids include, for example, target nucleic acid sequences from algae, bacteria, fungi, prions, viroids, viruses, or other parasitic pathogens. In some embodiments, the target nucleic acid sequence is from an upper respiratory virus, such as influenza, respiratory syncytial virus, adenovirus, coxsackievirus, parainfluenza, metapneumovirus, enterovirus, coronavirus, or streptococcus. In some aspects, provided herein are kits. In some embodiments, provided herein is a kit comprising a nucleic acid probe as described herein. In some embodiments, provided herein is a kit comprising a nucleic acid probe as described herein and one or more reagents for collection and / or storage of a sample and / or amplification of nucleic acid in a sample. For example, in some embodiments the kit additionally comprises sample collection tubes, preservatives, stabilizers, inhibitors (e.g. DNAse inhibitors, RNAse inhibitors, protease inhibitors, etc.), buffers, salts, dNTPS, enzymes, and the like. In some aspects, the kit comprises a nucleic acid probe as described herein, a polymerase, and a nicking enzyme. In some embodiments, the kit further comprises one or more calibrator or control reagents, such as positive or negative controls. In some embodiments, the kit comprises instructions for use of the kit, which may be in written form or may be accessible through an external means, including downloading from an internet site. EXAMPLES Example 1 Described herein is the development and use of single stranded oligonucleotide probes referred to herein as a Universal Hairpin Probes (UHPs). The UHPs provided herein detect the products of a nucleic acid amplification reaction. In some embodiments, the UHP comprises a universal hairpin, containing a fluorophore / fluorophore or fluorophore / quencher pair, with one signaling partner at the 5’ end and the other partner internally situated within an appropriate Förster radius. The UHP product recognition region extends from the 3’ end of the probe and can bind a sequence between or including the reactions’ amplification primers. In the absence of product, the UHP hairpin remains closed. Upon product binding, nicking enzyme and polymerase facilitate ALERE-40207.601 extension of a DNA strand complementary to the UHP. This strand extends through the hairpin, releasing the product, and separating the signaling partners, which results in a detectable change in fluorescence. As the bound product is released after extension through the hairpin, the UHP is neither incorporated into the product, nor does it significantly affect the underlying target amplification reaction. The UHP detects nucleic acid products rapidly, specifically, and with low background signal. The short product recognition region is readily changed without regard to secondary structure of the hairpin, these features streamline probe design, all but eliminating the need for costly probe optimization. UHP opening involves a molecular switch from a closed ssDNA UHP to an open dsDNA UHP. This unique feature has important implications for next generation detection schemes. Further, open UHPs exhibit high affinity for their complements and are not outcompeted by a secondary product, alleviating the requirement for one product to dominate the amplification reaction. Lastly, UHP synthesis is robust and insensitive to minor product deletions, reducing the risk of batch-to-batch variability. MATERIALS AND METHODS Oligonucleotides All oligonucleotides were purchased from IDT, Biosearch, or synthesized on solid phase with the phosphoramidite method using Dr. Oligo 48 Synthesizer. For in-house synthesized oligonucleotides, crude mixtures were analyzed on IPRP LC-MS and purified with a semi-prep C18 column. NEAR Reaction All reactions were performed in clean dead-air boxes. Unless otherwise noted, master mixes were prepared with a strand displacing DNA polymerase, nicking enzyme, reverse transcriptase (when testing RNA targets), dNTPs (Invitrogen), DTT, SYTO-82 (Invitrogen), and Isothermal Buffer with various salts and detergents. Template / Molecular Beacon or Template / UHP solutions were added to the 96 well plate followed by target RNA, diluted in RNA Storage Solution (Thermo) or DNA diluted in TE just prior to reaction. Reactions were run on a qPCR instrument for 10min at 56°C, followed by melting curve analysis from 60°C to 95°C. Data was analyzed using qPCR software and custom Python 3.0 scripts. ALERE-40207.601 IDNOW COVID, FluA, RSVA, RSVB, and StrepA IDNOW test bases containing either UHP or MB probes were run on the instruments according to standard protocols. Healthy control nasopharyngeal (NP) matrix was made by vigorously stirring NP swabs in prewarmed elution buffer, followed by addition of inactivated virus. These samples were immediately added to the test base and run. Mass Spectrometry All samples were analyzed on a Waters Xevo G2-XS mass spectrometer using DMBAA- ACN method over a range of 1000m / z to 2800m / z. MassLynx v4.1 software was used for data analysis. RESULTS As shown in FIGs. 1-2, UHP design is fast and novel. In the embodiments shown, UHPs have a universal hairpin with a 5’ fluorophore or quencher and an internal fluorophore or quencher pair. The product recognition region that binds to the sequence between the reaction’s amplification primers, here called the “spacer”, extends from the 3’ end of the hairpin. Various UHPs were shown to take ~5min to design without the need to balance stem and hybridization Tms. A range of UHP spacer regions between 11-16nt were tested, and all performed successfully. For many designs, a practical lower limit is ~8nt and the upper limit would be based on synthesizability of the probe, around ~30nt, although shorter or longer designs might be employed under special circumstances. An advantage of the probe is that the spacer region can be short and still retain specificity. The fluorophore should not be put next to a G base, as this could self-quench the fluorophore. The predicted universal hairpin melting temperature (Tm) should be between 60-70°C in the proper concentration of Mg2+. As shown in FIG. 3 and FIG. 4, UHPs have a unique mechanism of action. Both a nicking enzyme (NE) and polymerase are employed for UHP to extend, open, and be used for detection. By mass spectrometry, the fully extended UHP product comprising the fluorophore, quencher, universal hairpin, spacer region, and trailing template is found. UHP opening involves ALERE-40207.601 a molecular switch from a closed ssDNA UHP to an open and extended dsDNA UHP. This unique feature has important implications for next generation detection schemes. The trailing template, spacer, and universal hairpin in the open and extended UHP are longer, and thus stronger, than the product 1 and product 2 duplexes made in the underlying NEAR reaction. This means NEAR templates do not necessarily need to be skewed for them to work. The production of UHPs is robust in the face of synthesis failures and impurities. There are no 3’ modifications; as such, the UHP does not require a customized synthesis column. UHP impurities, including commonly seen deletions, do not affect the baseline, which is inherently low.

Claims

ALERE-40207.601 CLAIMS We claim:

1. A reaction mixture comprising: a) a polymerase; b) a nicking enzyme; and c) a nucleic acid probe comprising: a) a first region having a 3’ end and having a sequence complementary to at least a portion of a target nucleic acid; b) a second region 5’ of said first region having a first hairpin forming sequence; c) a third region 5’ of said second region having a loop structure; and d) a fourth region 5’ of said third region having a second hairpin forming sequence that hybridizes to said first hairpin forming sequence to form a hairpin but does not hybridize to said first region, said fourth region having a 5’ end.

2. The reaction mixture of claim 1, wherein said probe contains no detectable label, 3. The reaction mixture of claim 1, wherein said probe comprises a detectable label.

4. The reaction mixture of claim 3, wherein said probe comprises: a fluorophore attached to said second or said fourth region; and a quencher moiety attached to the other of said second or said fourth region.

5. The reaction mixture of claim 1, wherein when a target nucleic acid sequence binds to the first region of the nucleic acid probe, the nucleic acid probe undergoes a conformational change such that the position and size of the loop structure is modified, thereby producing a second loop structure that is smaller than the loop structure originally present within the third region of the nucleic acid probe.

6. The reaction mixture of claim 1, further comprising a target nucleic acid sequence.

7. A method of detecting a target nucleic acid in a sample, the method comprising forming a reaction mixture of any of claims 1 through 6 and detecting a target nucleic acid in the sample.

8. A method of detecting a target nucleic acid in a sample, the method comprising:ALERE-40207.601 a) contacting the sample with a nucleic acid probe, a polymerase, and a nicking enzyme; and b) detecting a target nucleic acid in the sample, wherein the nucleic acid probe comprises: a first region having a 3’ end and having a sequence complementary to at least a portion of a target nucleic acid; a second region 5’ of said first region having a first hairpin forming sequence; a third region 5’ of said second region having a loop structure; a fourth region 5’ of said third region having a second hairpin forming sequence that hybridizes to said first hairpin forming sequence to form a hairpin but does not hybridize to said first region, said fourth region having a 5’ end.

9. The method of claim 8, wherein the probe comprises a fluorophore attached to said second or said fourth region; and a quencher moiety attached to the other of said second or said fourth region.

10. The method of claim 8, wherein the target nucleic acid is pathogenic nucleic acid.

11. The method of claim 10, wherein the target nucleic acid is a gene transcript.

12. A kit comprising: a) a polymerase; b) a nicking enzyme; and c) a nucleic acid probe comprising: a first region having a 3’ end and having a sequence complementary to at least a portion of a target nucleic acid; a second region 5’ of said first region having a first hairpin forming sequence; a third region 5’ of said second region having a loop structure; and a fourth region 5’ of said third region having a second hairpin forming sequence that hybridizes to said first hairpin forming sequence to form a hairpin but does not hybridize to said first region, said fourth region having a 5’ end.

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