Nucleic Acid Detection Methods

Abstract

The present invention relates to a method for detecting nucleic acids of a defined sequence, and to a kit and apparatus for use in said method. The method utilizes restriction enzymes, polymerases, and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid. The amplification product is contacted with an oligonucleotide probe to produce a detector product. [Selection Diagram] Figure 1

Description

[Technical field]

[0001] (background) (Technical field) The present invention relates to a method for detecting nucleic acids of defined sequence, and to kits and devices for use in said method. [Background technology]

[0002] (Related Technology) Polymerase-based nucleic acid sequence amplification methods are widely used in the field of molecular diagnostics. The most established method, the polymerase chain reaction (PCR), typically involves two primers for each target sequence and uses temperature cycling in a process of cyclic exponential amplification to achieve primer annealing, extension by DNA polymerase, and denaturation of the newly synthesized DNA. The requirement for temperature cycling requires complex equipment, which limits the use of PCR-based methods in certain applications.

[0003] Strand Displacement Amplification (SDA) (EP0497272; US5455166; US5712124) was developed as an isothermal alternative to PCR that does not require temperature cycling to achieve annealing and denaturation of double-stranded DNA during polymerase amplification, but instead uses a restriction enzyme in combination with a strand-displacing polymerase to separate the two DNA strands.

[0004] In SDA, a restriction enzyme site at the 5' end of each primer is introduced into the amplification product in the presence of one or more alpha thiol nucleotides, and a restriction enzyme is used to nick the restriction site due to its ability to cleave only the strand with the unmodified hemiphosphorothioate form of its recognition site. A strand-displacing polymerase extends the 3' end of each nick and displaces the downstream DNA strand. Exponential amplification results in the pairing of sense and antisense reactants, where the strand displaced from the sense reactant serves as the target for the antisense reactant and vice versa. SDA typically takes more than an hour to perform, which greatly limits its potential for use in the field of clinical diagnostics. Furthermore, the requirement of separate processes for specific detection of the products after amplification and initiation of the reaction significantly increases the complexity of this method.

[0005] Maples et al. (WO2009 / 012246) then performed SDA using nicking enzymes, a subclass of restriction enzymes that can cleave only one of the two strands of DNA after binding to their specific double-stranded recognition sequence. They called this method the nicking and extension amplification reaction (NEAR). NEAR, which utilizes nicking enzymes rather than restriction enzymes, has also been used by others who have subsequently attempted to improve on this method using software-optimized primers (WO2014 / 164479) and through warm start or controlled temperature reduction (WO2018 / 002649). However, only a small number of nicking enzymes are available, making it more difficult to find an enzyme with the desired properties for a particular application.

[0006] A very important drawback of SDA (NEAR) using restriction or nicking enzymes is that the method produces double-stranded nucleic acid products and therefore does not provide an inherent process for efficient detection of the amplified signal. This significantly limits its usefulness, for example, in low-cost diagnostic devices. The double-stranded nature of the amplified products produced presents difficulties in combining the amplification method with signal detection, since hybridization-based detection cannot be performed without first separating the double strands. Therefore, more complex detection methods, such as molecular beacons and fluorophore / quencher probes, are required. This can complicate the assay protocol by requiring a separation process step, significantly reducing the potential for developing multiplex assays.

[0007] As a requirement to overcome the limitations of SDA, enhanced amplification methods for rapid, sensitive and specific detection of nucleic acid sequences are important.The present invention relates to a method for amplifying and detecting a target nucleic acid sequence that utilizes an additional oligonucleotide probe in addition to a primer pair with a 5' restriction site to produce a detector species that allows efficient signal detection. Summary of the Invention

[0008] (overview) The present invention relates to a method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) Purifying the amplification product of step a): i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; wherein hybridization of the first and second probes with the at least one species in the amplification product produces a detector species; and c) detecting the presence of the detector species produced in step b), the presence of the detector species being indicative of the presence of the target nucleic acid in the sample; The method includes:

[0009] An embodiment of the method is shown in FIG.

[0010] In various embodiments, in the presence of target nucleic acid, the methods rapidly produce many copies of detector species, ideally suitable for sensitive detection.

[0011] The present invention in various aspects is advantageous over known techniques because it includes rapid amplification without temperature cycling, in addition to providing an essential process for efficient detection of amplification products.

[0012] The method of the present invention overcomes a major drawback of SDA, including SDA with nicking enzymes (NEAR), in that SDA does not provide an intrinsic process for efficient detection of the amplification signal due to the double-stranded nature of the amplification product. The method overcomes this limitation by utilizing two additional oligonucleotide probes that hybridize with at least one species in the amplification product to facilitate its rapid and specific detection. The use of these two additional oligonucleotide probes provides the present invention with several additional advantages over known methods such as SDA. The first oligonucleotide probe is bound to a moiety that allows its detection and the second oligonucleotide probe is bound to a moiety that allows binding to a solid material or to the solid material. For example, in an embodiment of the present invention in which one of the oligonucleotide probes is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by a restriction enzyme and is contacted with the sample simultaneously with the performance of step a), unexpectedly no significant adverse inhibition of amplification is observed and a pre-detector species containing a single-stranded region is efficiently produced. This aspect of the invention is counterintuitive since one might assume that such blocked probes would result in asymmetric amplification biased towards the opposing amplicon strand contained in the predictor species, which in fact would be efficiently produced and ideally suited for efficient detection, since the exposed single-stranded regions would be readily available for hybridization of other oligonucleotide probes.

[0013] The inherent sample detection approach of the method is in stark contrast to previous attempts to overcome the critical limitations of SDA, including performing "asymmetric" amplification, for example by using unequal primer ratios to produce one amplicon strand in excess over the other. The method does not require asymmetric amplification and does not require the production of one amplicon strand in excess over the other, instead focusing on the production of detector species after hybridization of the first and second oligonucleotide probes. The inherent sample detection approach of the method, including the production of detector species, is ideally suited for combination with other detection methods, particularly nucleic acid lateral flow, for example by printing the second oligonucleotide probe on a lateral flow strip, which provides a simplified, rapid and low-cost means of performing the detection in step c). Combining the method with nucleic acid lateral flow also allows for an efficient multiplex system based on differential hybridization of multiple second oligonucleotide probes attached to discrete locations on a lateral flow strip, each with a different sequence designed for a different target nucleic acid sequence in the sample. In a further embodiment of the method, the efficiency of lateral flow detection is enhanced by the use of a single-stranded oligonucleotide as the moiety in the second oligonucleotide probe that allows binding to the solid material, and the reverse complementary sequence to said moiety is printed on the strip. The latter approach also allows the lateral flow strip to be optimized and manufactured as a single "universal" detection system across multiple target applications. This is because the sequence bound to the lateral flow strip can be defined and does not have to correspond to the sequence of the target nucleic acid. Thus, the essential requirement of two additional oligonucleotide probes in the method of the present invention provides many advantages over SDA, including SDA using nicking enzymes (NEAR).

[0014] The present invention is fundamentally different from SDA performed with nicking enzymes (NEAR) because it requires the use of a non-nicking restriction enzyme and one or more modified dNTPs, and has several additional advantages over such nicking enzyme-dependent methods. For example, there are many more non-nicking restriction enzymes available than nicking enzymes. This means that the restriction enzymes used in the method of the present invention can be selected from a large number of potential enzymes and identified as having superior properties for a given application, such as reaction temperature, buffer compatibility, stability, and reaction rate (sensitivity). Due to this important advantage of the method, the inventors can select restriction enzymes with lower optimum temperatures and faster rates than can be achieved with nicking enzymes. Such restriction enzymes are much more suitable for use in low-cost diagnostic devices. Furthermore, the requirement to use one or more modified dNTPs, in addition to having the restriction enzymes cleave only one strand of their restriction site, is an essential feature of the present invention that provides important advantages. For example, the use of certain dNTPs, such as α-thiol dNTPs, reduces the melting temperature (Tm) of DNA incorporating them. This means that the oligonucleotide primers and probes in this method have a higher affinity for hybridization with species in the amplification product than any competing complementary strands containing modified dNTPs produced during amplification. Furthermore, the lowering of the Tm of the amplification product as a result of the insertion of modified dNTP bases promotes the separation of double-stranded DNA species, thereby enhancing the amplification rate, lowering the optimum temperature, and improving sensitivity. Alternatively, the use of other modified dNTPs increases the Tm of the DNA into which they are incorporated, thereby offering further opportunities to tailor the implementation of the method for a given application.

[0015] Combining the many advantages of the present invention over SDA, the use of restriction enzymes or nicking enzymes (NEAR) provides the method utility in low-cost, disposable diagnostic devices with increased amplification rates and simple visualization of the amplified signal not possible with known methods.

[0016] Various embodiments of the above aspects of the invention as well as further aspects are described in more detail below. [Brief description of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 is a schematic representation of a method according to one embodiment of the present invention. [Diagram 2] Schematic representation of a method, wherein the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with a sample in step a). [Diagram 3] Schematic representation of steps b) and c) of the method, wherein the moiety of the second oligonucleotide probe enabling binding to a solid material is a single-stranded oligonucleotide. [Figure 4] Schematic representation of part of step a) of the method, in which in step a) the sample is further contacted with a third and a fourth oligonucleotide primer. [Diagram 5] A second oligonucleotide probe is attached to a solid material, a nitrocellulose lateral flow strip, in accordance with the method of the present invention (see Example 1). [Figure 6] Figures 6A and 6B. Implementation of a method (see Example 2) in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and in which the probe is contacted with a sample in step a). [Figure 7] Figures 7A, 7B, 7C, and 7D. Implementation of a method in which the presence of two or more different target nucleic acids of defined sequence is detected in the same sample (see Example 3). [Figure 8] A practice of the method wherein the first and second hybridization sequences in the target nucleic acid are separated by 5 bases (see Example 4). [Figure 9]Implementation of a method in which the moiety that enables binding of the second oligonucleotide probe to a solid material is an antigen and the corresponding antibody is bound to a solid surface, a nitrocellulose lateral flow strip (see Example 5). [Figure 10] 10A and 10B. An implementation of a method in which the moiety that enables binding of a second oligonucleotide probe to a solid material is a single-stranded oligonucleotide that contains four repeating copies of a three-base DNA sequence motif, and the reverse complement of the single-stranded oligonucleotide sequence is bound to the solid material (see Example 6). [Figure 11] Use of RNA virus detection methods in clinical specimens (see Example 7). [Figure 12] 12A and 12B. Performance of the method at different temperatures (see Example 8). [Figure 13] Figures 13A and 13B. Implementation of a method in which a target nucleic acid is obtained from double-stranded DNA by strand invasion (see Example 9). [Figure 14] 14A and 14B. Comparative implementation of the method of the present invention with known methods (see Example 10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] (Detailed Description) The present invention provides a method for detecting the presence of a single-stranded target nucleic acid of a defined sequence in a sample. The target nucleic acid can be single-stranded DNA, including single-stranded DNA derived from double-stranded DNA after dissociation of the double strand in the sample, for example, by heat denaturation or through the strand displacement activity of a polymerase, or single-stranded DNA derived from RNA, for example, by the activity of reverse transcriptase, or single-stranded DNA derived from double-stranded DNA, for example, by the use of a nuclease, such as a restriction endonuclease or exonuclease III, or single-stranded DNA derived from an RNA / DNA hybrid, for example, through an enzyme, such as RNase H. The target nucleic acid can be single-stranded DNA derived from DNA in the sample by a DNA polymerase, a helicase, or a recombinase. The single-stranded site in the double-stranded DNA can be sufficiently exposed for hybridization and extension of the first oligonucleotide primer to initiate the method, for example, by "strand invasion", where a transient opening of one or more DNA base pairs in the double-stranded DNA occurs sufficiently to allow hybridization of the first oligonucleotide primer and extension of the 3' hydroxyl, or by spontaneous opening of DNA base pairs, transitional conversion to Hoogsteen pairs, or by productive nicking of DNA by restriction enzymes or thermochemical approaches. The target nucleic acid can be single-stranded RNA, including single-stranded RNA derived from double-stranded RNA in the sample after dissociation of the two strands, for example, by heat denaturation, or single-stranded RNA derived from double-stranded DNA, for example, by transcription.

[0019] The method comprises, in step a) subjecting the sample to: (i) a first oligonucleotide primer and a second oligonucleotide primer, the first primer comprising, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprising, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; (ii) strand-displacing DNA polymerase; (iii) dNTPs; (iv) one or more modified dNTPs; (v) a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by a DNA polymerase using the one or more modified dNTPs; and (vi) contacting with a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site if the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by a DNA polymerase using the one or more modified dNTPs.

[0020] When the target nucleic acid to be detected in the sample is double-stranded, either strand can be considered as the single-stranded target nucleic acid of this method.This is because one of the two oligonucleotide primers can hybridize with one strand, and the other oligonucleotide primer can hybridize with the other strand.Typically, the oligonucleotide primer used in this method is a DNA primer that forms a hybrid duplex with DNA or RNA target, comprising double-stranded DNA or both strands of RNA and DNA.However, primers that comprise other nucleic acids, such as non-natural bases and / or alternative backbone structures, can also be used.

[0021] In the presence of a target nucleic acid, the first oligonucleotide primer hybridizes to a first hybridization sequence in the target nucleic acid. After said hybridization, the 3' hydroxyl group of the first primer is extended by a strand-displacing DNA polymerase, or optionally, if the target nucleic acid is RNA, a reverse transcriptase (e.g., M-MuLV), to produce a double-stranded species containing the extended first primer and the target nucleic acid (see FIG. 1). The strand-displacing DNA polymerase or, if present, a reverse transcriptase uses dNTPs and one or more modified dNTPs in said extension. One strand of the restriction enzyme recognition sequence and the cleavage site at the 5' end of the first primer typically do not hybridize, since their reverse complementary sequences are generally not present in the target nucleic acid sequence. Thus, the first primer is generally used to introduce one strand of the restriction enzyme recognition sequence and the cleavage site into the subsequent amplification product species. After the extension of the first primer, "target removal" occurs. Target removal renders the extended first primer species accessible for hybridization with the reverse complement of the second hybridization sequence of the second oligonucleotide primer. If the target nucleic acid is RNA, target removal can be achieved, for example, by RNase H degradation of the RNA, by the RNase H activity of the reverse transcriptase if present, or by separate addition of this enzyme. Alternatively, if the target nucleic acid is single-stranded DNA containing a single-stranded region in the double-stranded DNA, target removal can be achieved by strand displacement with an additional upstream primer or bump primer. Alternatively, such target removal can occur after spontaneous dissociation, especially when only short extension products are produced from a given target nucleic acid molecule, or target removal can occur by strand invasion, where a transient opening of one or more DNA base pairs in the double-stranded extended first primer species occurs sufficient to allow hybridization of the strand-displaced second oligonucleotide primer and extension of the 3' hydroxyl.

[0022] After hybridization of the second oligonucleotide primer with the reverse complement of the second hybridization sequence, a strand-displacing DNA polymerase extends the 3' hydroxyl of the primer with dNTPs and one or more modified dNTPs. A double-stranded restriction recognition sequence and cleavage site for the first restriction enzyme is formed, with one or more modified dNTP bases incorporated into the reverse complement, acting to block cleavage of the strand by the first restriction enzyme. The first restriction enzyme recognizes its recognition sequence and cleaves only the first primer strand at the cleavage site. This creates a 3' hydroxyl that is extended by the strand-displacing DNA polymerase with dNTPs and one or more modified dNTPs, displacing the first primer strand. A double-stranded restriction recognition sequence and cleavage site for the second restriction enzyme is formed, with one or more modified dNTP bases incorporated into the reverse complement, acting to block cleavage of the strand by the second restriction enzyme. Thus, a double-stranded species is produced in which the two primer sequences are juxtaposed, and there are partially blocked recognition sites for the first and second restriction enzymes. Subsequent cleavage of the first primer strand by a first restriction enzyme and cleavage of the second primer strand by a second restriction enzyme occurs to produce two double-stranded species, one containing the first primer sequence and the other containing the second primer sequence. Sequential cleavage and displacement of the first and second primer strands then occurs in a cyclic amplification process, where the displaced first primer strand acts as a target for the second primer and the displaced second primer strand acts as a target for the first primer.

[0023] In the presence of target nucleic acid, amplification products are produced without any need for temperature cycling.

[0024] An essential aspect of the invention is that instead of directly detecting the amplification product of step a), a detector species is produced after specific hybridization of the first and second oligonucleotide probes with at least one species in the amplification product of both: the first oligonucleotide probe is bound to a moiety that allows its detection and hybridizes with a first single-stranded detection sequence of said at least one species; the second oligonucleotide probe is bound to a moiety that allows binding to the solid material or to said solid material and hybridizes with a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of said at least one species.

[0025] It will be apparent to one skilled in the art by reference to Figure 1 that the amplification product may include several different species, including species that consist of full length or partial or reverse complementary sequences of both the first and second primers, which may be separated by target derived sequences, provided that the primers that bind to the first and second hybridization sequences in the target nucleic acid are separated by one or more bases. It will further be apparent that any of the species may be selected to hybridize with the first and second oligonucleotide probes to form detector species.

[0026] The detector species produced in step b) is detected in step c), where the presence of the detector species indicates the presence of the target nucleic acid in the sample.

[0027] By utilizing two oligonucleotide probes, one for detection and one for binding to the solid material, the method of the invention provides rapid and efficient signal detection, overcoming the need for more complex secondary detection methods, such as by nucleic acid lateral flow, and providing efficient visualization of the signal produced in the presence of the target.

[0028] The method of the invention may be carried out, wherein one of said first and second oligonucleotide probes is blocked at the 3' end for extension by a strand displacing DNA polymerase and cannot be cleaved by a first or second restriction enzyme. Thus, according to a further embodiment, the invention provides a method for detecting the presence of a single stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) subjecting the amplification product of step a) to: i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and a detector species is produced by hybridization of the first and second probes with the at least one species in the amplification product; and c) detecting the presence of the detector species produced in step b), the presence of the detector species indicating the presence of the target nucleic acid in the sample.

[0029] In one embodiment, said one blocked oligonucleotide probe cannot be cleaved by said first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate bonds. In a further embodiment, said one blocked oligonucleotide probe is contacted with the sample simultaneously with, i.e. during, the performance of step a) so that it is present during the production of an amplification product in the presence of a target nucleic acid. Thus, according to a further embodiment, the present invention provides a method for detecting the presence of a single-stranded target nucleic acid of a defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) Purifying the amplification product of step a): i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and is contacted with the sample simultaneously with performing step a), where a detector species is produced by hybridization of the first and second probes with the at least one species in the amplification product; and c) detecting the presence of the detector species produced in step b), the presence of the detector species indicating the presence of the target nucleic acid in the sample.

[0030] For example, in the embodiment shown in FIG. 2, a first oligonucleotide probe is blocked and hybridizes to a first single-stranded detection sequence of at least one species in the amplification product to form a predictor species comprising a single-stranded region. The at least one species can be extended by a strand-displacing DNA polymerase that extends its 3' hydroxyl group, thus further stabilizing the predictor species. Thus, in the embodiment, the blocked oligonucleotide probe comprises an additional region, and the 3' end of the species in the amplification product to which the blocked oligonucleotide probe hybridizes can be extended by the strand-displacing DNA polymerase. As shown in FIG. 2, a "stable predictor species" is produced. Those skilled in the art will recognize that this additional predictor species stabilizing region in the blocked oligonucleotide probe is upstream of the region that hybridizes to the first or second single-stranded detection sequence in the at least one species in the amplification product. In embodiments using blocked oligonucleotide probes, the hybridization sequence of the blocked oligonucleotide probe and the appropriate concentration of primers can be optimized so that a certain ratio of related species produced in the amplification product hybridizes to the blocked oligonucleotide probe in each cycle, and remaining copies of such species remain available to participate in the cyclic amplification process. The oligonucleotide probe is blocked from extension, for example, by using a 3' phosphate modification, and in this embodiment, is attached to a moiety that allows its detection, for example a 5' biotin modification. Alternatively, a single 3' modification can be used to block extension and as a moiety that allows its detection. A variety of other modifications can be used to block the 3' end of the oligonucleotide, such as a C-3 spacer, or a mismatched base can be used instead. The predictor species is ideally suited for efficient detection, since the exposed single-stranded region remains readily available for hybridization with a second oligonucleotide probe. The second oligonucleotide probe can be attached to the nitrocellulose surface of a nucleic acid lateral flow strip.This allows sequence-specific hybridization to occur easily as the detector species flows through it, localizing the detector species to defined locations on the strip. Dyes that bind to detection moieties, such as streptavidin-conjugated carbon, gold, or polystyrene particles, that may be present in the conjugate pad of the nucleic acid lateral flow strip or during the amplification reaction, provide rapid color-based visualization of the presence of the detector species produced in the presence of the target nucleic acid.

[0031] In another embodiment, it is said second oligonucleotide probe, whose extension by said strand-displacing DNA polymerase is blocked at its 3' end, and which cannot be cleaved by said first or second restriction enzyme, and the sample is contacted at the same time as the step a) is performed. The second oligonucleotide probe can be bound to a solid material, such as the surface of an electrochemical probe, a 96-well plate, a bead, or an array surface, or bound to a moiety that allows its binding to a solid material, before contacting with the sample. At least one species of a certain ratio produced during the amplification hybridizes to the second oligonucleotide probe after its production, rather than hybridizing to the associated reaction primer that further participates in the cyclic amplification process. After hybridization with the second oligonucleotide probe, said species is extended by a polymerase on the oligonucleotide probe to produce a stable predictor species. Also, the first oligonucleotide probe and the detection moiety can be contacted with the sample at the same time as the step a) is performed, and become localized to said surface at the site of the second oligonucleotide probe. Detecting the accumulation of the detection moiety at the site during the amplification process provides a real-time signal that provides quantification of the number of copies of the target nucleic acid present in the sample. Thus, according to an embodiment of the invention, two or more of steps a), b), and c) are performed simultaneously.

[0032] In carrying out these embodiments, in which one of the first and second oligonucleotide probes is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and is contacted with the sample simultaneously with carrying out step a), the inventors have not observed any significant inhibition of the amplification rate, indicating that the predictor species accumulates in real time without disrupting the optimal cyclic amplification process. This is in contrast to attempts to operate asymmetric SDA by utilizing unequal primer ratios with the goal of producing one amplicon strand in excess over the other. Rather than seeking to use blocked oligonucleotide probes to remove one amplicon strand from the reaction, thereby increasing the ratio of the other strand, the present invention focuses on the production and detection of detector species using blocked probes to promote exposure of single-stranded regions during the amplification process. Thus, not only did the inventors not observe any inhibitory effect on the amplification process in the above embodiments, but in one embodiment, the inventors observed an unexpected enhancement in the generated signal corresponding to at least a 100-fold increase in the amount of detector species (see Example 2) (Figure 6).

[0033] Moreover, said embodiment of the method of the invention, in which one of the first and second oligonucleotide probes is blocked at its 3' end from extension by DNA polymerase and cannot be cleaved by the first or second restriction enzyme, is contacted with the sample simultaneously with the performance of step a), a fundamental advantage over reported attempts to integrate NEAR with nucleic acid lateral flow in a multi-step process without blocking the probe. For example, in WO2014 / 164479, a long incubation time of 30 minutes at 48°C was required to visualize the amplification products using nucleic acid lateral flow, which represents a major barrier to the use of the method in point-of-care diagnostic devices, especially low-cost or disposable devices. In stark contrast, in the method of the invention, comparable amplification is readily performed within 5 minutes at lower incubation temperatures, e.g., 40-45°C. Moreover, in a direct comparative study (see Example 10), the method of the present invention shows unexpected and significantly superior speed compared to the prior art method (WO2014 / 164479) which results from the combination of the use of a non-nicking restriction enzyme, the use of modified dNTP bases, and the use of said blocked oligonucleotide probes.

[0034] It should also be appreciated that the other of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme described above.

[0035] An essential aspect of the method is the use of one or more restriction enzymes that are not nicking enzymes but are capable of recognizing its recognition sequence and cleaving only one strand of its cleavage site, when said recognition sequence and cleavage site are double stranded and cleavage of the reverse complementary strand is prevented by the presence of one or more modifications incorporated into said reverse complementary strand by a strand displacing DNA polymerase using one or more modified dNTPs, e.g. dNTPs that confer nuclease resistance after incorporation by the polymerase.

[0036] "Restriction enzymes" [or "restriction endonucleases"] are a broad class of enzymes that cleave one or more phosphodiester bonds in one or both strands of a double-stranded nucleic acid molecule at a specific cleavage site after binding to a specific recognition sequence. A large number of restriction enzymes are available, with over 3,000 described and over 600 commercially available, covering a wide variety of different physicochemical properties and recognition sequence specificities.

[0037] "Nicking enzymes" [or "nicking endonucleases"] are a specific subclass of restriction enzymes that, after binding to a specific recognition sequence, can cleave only one strand of a double-stranded nucleic acid molecule at a specific cleavage site, leaving the other strand intact. Only a few (c.10) nicking enzymes are available, both natural and engineered. Nicking enzymes include the bottom strand cutters Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, and Nb.BtsI and the top strand cutters Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.

[0038] Although the non-nicking restriction enzymes used exclusively in the methods of the present invention can cleave both strands of double-stranded nucleic acid, they can also cleave or nick only one strand of their double-stranded DNA cleavage site under certain circumstances after binding to their recognition sequence. This can be achieved in several ways. Of particular relevance to the present methods, this can be achieved when one of the strands in the double-stranded nucleic acid at the cleavage site is rendered uncleavable because one strand of the double-stranded nucleic acid target site is modified such that the phosphodiester bond at the cleavage site on one strand is protected with a nuclease-resistant modification, such as phosphorothioate (PTO), boranophosphate, methylphosphate, or peptide-internucleotide bond. Certain modified internucleotide bonds, such as PTO bonds, can be provided in oligonucleotide probes and primers by chemical synthesis or can be incorporated into double-stranded nucleic acid by polymerase, for example, using one or more α-thiol modified deoxynucleotides. Thus, in one embodiment, the one or more modified dNTPs are α-thiol modified dNTPs. Typically, the S isomer is utilized, which is incorporated more efficiently and confers nuclease resistance.

[0039] Due to the large number of non-nicking restriction enzymes available, a wide variety of enzymes with different properties are available to screen for desired performance characteristics, such as temperature profile, speed, buffer compatibility, polymerase cross-compatibility, recognition sequence, thermostability, manufacturability, etc., for use in a method for a given application. In contrast, the fact that only a few nicking enzymes are available limits the potential of prior art methods that use nicking enzymes, which may result in, for example, lower reaction rates (sensitivity, time to result) and higher reaction temperatures. The non-nicking restriction enzymes selected for use in the method may be natural or engineered enzymes.

[0040] In selecting a restriction enzyme that is not a nicking enzyme for use in this method, those skilled in the art will recognize that it is necessary to identify an enzyme with a suitable cleavage site to ensure that the modification is incorporated at the appropriate position to prevent cleavage of the relevant strand and not the other strand.For example, in embodiments using modified dNTPs, such as α-thiol dNTPs, it may be preferable to select a restriction enzyme with a cleavage site outside of its recognition sequence, such as an asymmetric restriction enzyme with a non-palindromic recognition sequence, to provide sufficient flexibility in primer positioning so that the target nucleic acid sequence contains a modified nucleotide base at the appropriate position to prevent cleavage of the relevant strand after incorporation.For example, when using α-thiol dATP, the reverse complementary sequence of the restriction enzyme cleavage site of the relevant oligonucleotide primer contains an adenosine base downstream of the cleavage site in the reverse complementary strand, but does not contain an adenosine base downstream of the cleavage site in the primer sequence, to ensure that the primer is properly cleaved in the implementation of the method.Therefore, an asymmetric restriction enzyme with a non-palindromic recognition sequence that ideally cleaves outside its recognition sequence is suitable for use in the present invention. Restriction enzymes that recognize partial or degenerate palindromic sequences that cut within their recognition sites can also be used. Nuclease-resistant nucleotide binding modifications, such as PTO, can be used to prevent the cleavage of either strand by a wide variety of commercially available double-stranded cleavage agents from a variety of different classes. Such classes of double-stranded cleavage agents include type IIS and type IIG restriction enzymes that have both partial or degenerate palindromic and asymmetric restriction enzyme sequences to enable their use in the methods of the present invention.

[0041] Restriction enzymes are typically utilized in this method in amounts of 0.1-100 units, where 1 unit is defined as the amount of agent required to digest 1 μg T7 DNA in 1 hour at a given temperature (e.g., 37° C.) in a total reaction volume of 50 μl. However, the amount will vary depending on several factors, such as the activity of the selected enzyme, the concentration and form of the enzyme, the expected concentration of the target nucleic acid, the reaction volume, the concentration of primers, the reaction temperature, etc., and should not be considered limiting in any way. One of skill in the art will appreciate that the restriction enzymes utilized in this method require suitable buffers and salts, such as divalent metal ions, for effective and efficient function, pH control, and enzyme stabilization.

[0042] In one embodiment, the first and second restriction enzymes are the same restriction enzyme.By using only a single restriction enzyme, the method is simplified in many ways.For example, only one enzyme that is compatible with other reaction components needs to be identified, optimized, manufactured and stabilized for the implementation of the method.In addition, the use of a single restriction enzyme simplifies the design of oligonucleotide primers and supports the symmetry of the amplification process.

[0043] In the method, the restriction enzymes cleave only one strand of the nucleic acid duplex, and therefore after cleavage, they present an exposed 3' hydroxyl group that can act as an efficient polymerase priming site. Polymerases are enzymes that synthesize nucleic acid strands or polymers by extending primers and using base pairing interactions to generate reverse complementary copies of DNA or RNA template strands. Polymerases with strand displacement ability are utilized in the implementation of the method, as they appropriately displace strands to affect the amplification process. The term "strand displacement" refers to the ability of a polymerase to displace downstream DNA that it encounters during synthesis. Various polymerases with strand displacement ability that operate at different temperatures have been characterized and are commercially available. For example, Phi29 polymerase has a very strong ability to displace strands. Polymerases from Bacillus species, such as Bst DNA polymerase large fragment, typically exhibit high strand displacement activity and are well suited for use in the implementation of the method. The E. coli Klenow fragment (exo-) is another widely used strand-displacing polymerase. Strand-displacing polymerases, such as KlenTaq, can be easily engineered, such as by cloning only the relevant active polymerase domain of the endogenous enzyme and knocking out any exonuclease activity. For the implementation of the method, where the single-stranded target nucleic acid is RNA, an RNA-dependent DNA synthesis (reverse transcriptase) activity is also required. This activity can be performed by the strand-displacing polymerase and / or by a separate additional reverse transcriptase in step a), such as M-MuLV or AMV.

[0044] Polymerases are typically utilized in appropriate amounts optimized for the relevant steps of the method depending on the enzyme, reagent concentrations, and desired reaction temperature. For example, 0.1 to 100 units of Bacillus polymerase can be used, with one unit defined as the amount of enzyme that incorporates 25 nmol of dNTPs into acid-insoluble material in 30 minutes at 65° C. However, the amount depends on several factors, such as the polymerase activity, its concentration and form, the expected concentration of the target nucleic acid, the reaction volume, the number and concentration of oligonucleotide primers, and the reaction temperature, and should not be considered limiting in any way.

[0045] Those skilled in the art will know that dNTP monomers are required for a polymerase to have polymerase activity, and that polymerases require an appropriate buffer, including components such as buffer salts, divalent ions, and stabilizers. In addition, one or more modified dNTPs are used in this method to prevent cleavage of the reverse complementary strand of the primer after incorporation by a strand-displacing polymerase. Typically, when a single modified dNTP is used, the dNTP used in this method lacks the corresponding base. For example, in an embodiment where the modified dNTP is α-thiol dATP, the dNTP contains only dTTP, dCTP, and dGTP, but does not contain dATP. Removal of the corresponding natural dNTP bases ensures that all required bottom strand cleavage sites in the reverse complementary sequence of the primer are blocked, since only modified bases are available for incorporation by the polymerase. However, complete or partial removal of the corresponding natural dNTP bases is not required. dNTPs may typically be used in this method at concentrations comparable to those utilized in other polymerase methods, e.g., in the range of 10 micromolar to 1 millimolar, although dNTP concentrations may be optimized for any given enzyme and reagent for this method with the goal of maximizing activity and minimizing synthesis from 1 to avoid the generation of background signal. Given that a particular polymerase may exhibit a lower rate of incorporation of one or more modified dNTP bases, one or more modified bases may be used in this method at a higher relative concentration than the unmodified dNTP, e.g., 5-fold higher, although this should be considered non-limiting.

[0046] The use of one or more modified dNTPs is an essential feature of the present invention that provides important advantages in addition to allowing restriction enzymes to cleave only one strand of their restriction site. For example, the use of certain modified dNTPs, such as alpha-thiol dNTPs, reduces the melting temperature (Tm) of DNA incorporating them. This means that the oligonucleotide primers and probes used in this method have a higher affinity for hybridization to species in the amplification product than any competing modified dNTP complement produced during amplification. This important feature enhances the rate of amplification, for example, by providing a lower Tm for hybridization when one of the displaced strands hybridizes to its reverse complement to produce a "non-productive" end species, which dissociates more easily than "productive" hybridization of the displaced strand with additional primers. It has been reported that phosphorothioate internucleotidic linkages can lower the Tm (the temperature at which exactly half of the single strands of a duplex hybridize) by 1-3°C per addition, significantly altering physicochemical properties. The inventors have also observed that the presence of phosphorothioate nucleotide bonds in the DNA sequence enhances the strand displacement rate. Moreover, the oligonucleotide probes used in this method, whether they are contacted with the sample simultaneously with the implementation of step a) or after step a), have a higher affinity to the species in the amplification product than any competing modified species, and therefore may preferentially hybridize or even displace the hybridized strand, promoting the production of detector species. The reduced Tm and enhanced displacement of the amplification product species as a result of the modified internucleotide bonds contained therein serve to radically enhance the rate of this method and reduce the temperature required for rapid amplification to occur.

[0047] In addition to the rate enhancement by using one or more modified nucleotides, the specificity of hybridization of oligonucleotide primers and probes of this method is also enhanced. Typically, assuming that all bases of one particular nucleotide are substituted in the amplification product, the hybridization sites of primers and probes typically contain modified bases, and the reduced Tm caused by phosphorothioate internucleotide bonds means that sequence mismatches due to, for example, non-specific hybridization are less likely to be tolerated.

[0048] Thus, the essential feature of the method of the invention of one or more modified dNTPs enhances both the sensitivity and specificity of the amplification, providing a fundamental advantage that is in stark contrast to known methods that do not require such modified nucleotides, such as NEAR (WO2009 / 012246), which includes variations on NEAR using software-optimized primers (WO2014 / 164479) or warm start or controlled temperature reduction (WO2018 / 002649).

[0049] There are several different modified dNTPs, modified dNTPs that confer nuclease resistance after their incorporation by polymerase, and can be used in the method to achieve resistance to restriction enzyme cleavage and, in embodiments, other features that enhance the performance of the method for a given application. In addition to α-thiol dNTPs that provide nuclease resistance and reduced Tm, modified dNTPs that have been reported to have the potential for polymerase incorporation and confer nuclease resistance include equivalent nucleotide derivatives such as borano derivatives, 2'-O-methyl (2'OMe) modified bases and 2'-fluoro bases. Other modified dNTPs or equivalent compounds that may be incorporated by the polymerase and used in embodiments of the method to enhance certain properties of the method include those that decrease binding affinity, such as inosine-5'-triphosphate or 2'-deoxyzebularine-5'-triphosphate, those that increase binding specificity, such as 5-methyl-2'-deoxycytidine-5'-triphosphate, or 5-[(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine-5'-triphosphate, and those that enhance synthesis of GC-rich regions, such as 7-deaza-dGTP. Certain modifications can increase the Tm, providing further potential for controlling hybridization events in embodiments of the method.

[0050] Steps a), b) and c) can be carried out over a wide range of temperatures. The optimum temperature for each step is determined by the optimum temperature of the associated polymerase and restriction enzyme, as well as the melting temperature of the hybridized region of the oligonucleotide primer. Notably, the method does not use temperature cycling in step a). Moreover, the amplification step a) does not require any controlled increase or decrease in temperature, hot or warm start, preheating, or controlled temperature reduction. The method allows the steps to be carried out over a wide temperature range, for example, from 15°C to 60°C, such as from 20°C to 60°C or from 15°C to 45°C. According to an embodiment, step a) is carried out at a temperature below or about 50°C. Considering the wide variety of restriction enzymes that are not nicking enzymes available for use in the method, it is possible to select a restriction enzyme that has a rapid rate at relatively low temperatures compared to alternative methods that use nicking enzymes. The use of one or more modified nucleotides also reduces the temperature required for amplification. In addition to having the potential to lower the optimal temperature profile compared to known methods, the method of the present invention can be performed in an exceptionally wide temperature range. Such features make the method very attractive for use in low-cost diagnostic devices, where heating control imposes complex physical constraints that increase the cost of goods for such devices to the point that disposable or instrument-free devices are not commercially viable. For example, several assays have been developed using methods that can perform rapid detection of target nucleic acids at ambient temperature or approximately 37°C. Thus, in a further embodiment, step a) is performed at a temperature of less than or about 45°C. To simplify the user steps and reduce the overall time to result, it may be preferable to start the method at a temperature lower than the target temperature. Thus, in a further embodiment of the method, the temperature of step a) is increased during amplification. For example, the temperature of the method starts at ambient temperature, e.g., 20°C, and is increased over a period of time, e.g., 2 minutes, to reach a final temperature, e.g., approximately 45°C or 50°C. In one embodiment, the temperature is increased during the performance of step a), such as increasing from an initial ambient temperature, for example in the range of 15-30°C, to a temperature in the range of 40-50°C.

[0051] The low temperature potential and versatility of the method of the invention means that, in contrast to known methods, the method of the invention is compatible with the conditions required for a variety of other assays, such as immunoassays or enzymatic assays for detecting other biomarkers, such as proteins or small molecules. Thus, the method can be used, for example, for the simultaneous detection of both a nucleic acid of interest and a protein or small molecule in a sample. The components required to perform the method, including a non-nicking restriction enzyme, a strand-displacing DNA polymerase, an oligonucleotide primer, an oligonucleotide probe, dNTPs and one or more modified dNTPs, can be lyophilized or freeze-dried for stable storage, and the reaction can be subsequently triggered by rehydration, for example upon addition of a sample. Such lyophilization or freeze-drying for stable storage typically requires the addition of one or more excipients, such as trehalose, before drying the components. A wide variety of such excipients and stabilizers for lyophilization or freeze-drying are known and available for testing to identify suitable compositions of the components required to perform the method.

[0052] It will be apparent to one of skill in the art that the methods of the present invention are polymerase-based amplification methods and may be enhanced by the addition of one or more additives that have been shown to enhance PCR or other polymerase-based amplification methods. Such additives include, but are not limited to, tetrahydrothiophene 1-oxide, L-lysine free base, L-arginine, glycine, histidine, 5-aminovaleric acid, 1,5-diamino-2-methylpentane, N,N'-diisopropylethylenediamine, tetramethylenediamine (TEMED), tetramethylammonium chloride, tetramethylammonium oxylate, methylsulfonacetamide, hexadecyltrimethylammonium bromide, betaine aldehyde, tetraethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, tetrabutylammonium chloride, tetrapropylammonium chloride, formamide, dimethylformamide (DMF), N-methylformamide, N-methylacetamide, N,N-dimethylacetamide, L-threonine, N,N-dimethylethylenediamine, 2-pyrrolidone, HEP (N-hydroxyethylpyrrolidone), NMP. (N-methylpyrrolidone), and 1-methyl, 1-cyclohexyl-2-pyrrolidone (pyrrolidinone), δ-valerolactam, N-methylsuccinimide, 1-formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose, glycerol, Tween-20, DMSO, betaine, and BSA.

[0053] Our research has revealed that the method is effective over a wide range of target nucleic acid levels, including detection of very low copy numbers, even single copy numbers. The oligonucleotide primers are typically provided in large excess to the target nucleic acid. Typically, the concentration of each primer is in the range of 10-200 nM, but should not be considered limiting. Higher primer concentrations may enhance the efficiency of hybridization and thus increase the reaction rate. However, non-specific background effects such as primer dimers may also be observed at higher concentrations, so the concentrations of the first and second oligonucleotide primers form part of the optimization process for any given assay utilizing this method. In one embodiment, the first and second oligonucleotide primers are provided at the same concentration. In another embodiment, one of the first and second oligonucleotide primers is provided in excess of the other primer. Due to the natural symmetry of the cyclic amplification process, one primer is provided in excess of the other, although in certain circumstances, it may be used to reduce non-specific background signals of the method and / or to enhance the ability of the first and second oligonucleotide probes to hybridize and produce a detector species, although in embodiments the reaction rate may be slowed. It is desirable for both primers to be present at non-limiting levels before sufficient detector species is produced to be detected by the chosen detection means.

[0054] There are several considerations in the design of oligonucleotide primers to enhance the performance of this method. The first and second oligonucleotide primers must each contain a restriction enzyme recognition sequence and cleavage site in the 5' to 3' direction, as well as one strand of a hybridization region, which can hybridize with a first hybridization region in the target nucleic acid in the case of the first primer, and with the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid in the case of the second primer. Thus, a pair of primers is designed to amplify a region of the target nucleic acid. The restriction enzyme recognition sequence of the primer is typically not present in the target nucleic acid sequence, and is introduced into the amplicon after forming an overhang during the first hybridization event (see FIG. 1). When using an asymmetric restriction enzyme, the cleavage site is typically downstream of the recognition sequence, and thus can be optionally present in the hybridization sequence of the primer.

[0055] The oligonucleotide primer is designed such that after its cleavage in this method, the sequence 5' of the cleavage site continues to hybridize with its reverse complement under the desired reaction conditions, forming an upstream primer with a melting temperature (Tm) sufficient to displace the strand downstream of the cleavage site after extension of the 3' hydroxyl group by a strand-displacing DNA polymerase. Thus, an additional "stabilizing" region may be included at the 5' end of the oligonucleotide primer. Its optimal length is determined by other factors such as the location of the cleavage site relative to the recognition sequence of the relevant restriction enzyme and the temperature utilized for amplification in step a). Thus, in one embodiment, the first and / or second oligonucleotide primer comprises a stabilizing sequence upstream of the restriction enzyme recognition sequence and the cleavage site, e.g., at the 5' end, and e.g., 5 or 6 bases long.

[0056] In primer design, the sequence and length of each hybridization region must be defined to allow optimal sequence-specific hybridization and strand displacement to ensure specific and sensitive amplification in this method. The positioning of the primer in the genome of the target nucleic acid to be detected, such as a virus or bacterial pathogen, defines the sequence of the hybridization region of the primer in various ways, so that the sensitivity and specificity of the amplification can be optimized and primers with compatibility with the oligonucleotide probe can be selected. Different primer pairs can therefore be screened to identify the optimal sequence and positioning to enhance the performance of this method. Typically, the length of the hybridization region of the primer is designed so that its theoretical Tm allows efficient hybridization at the desired reaction temperature, but at the same time is easily displaced after cleavage. During primer design, the theoretical Tm of the hybridization sequence and the sequence of the displaced strand are considered in the context of the expected reaction temperature and the selected restriction enzyme. This is balanced against the theoretical improvement towards sequence-derived binding specificity that may occur with longer sequence lengths. Various studies by the inventors have shown that there is considerable versatility in designing primers for effective use in this method. In one embodiment, the hybridizing region of the first and / or second oligonucleotide primer is 6-30 bases long, for example 9-16 bases long. In further embodiments, modifications such as unnatural bases and alternative internucleotide linkages, or abasic sites, can be utilized in the hybridizing region of the primer to refine their properties and the operation of the method for specific applications. For example, modifications that enhance Tm, such as PNA, LNA, G-clamp, can allow for shorter and more specific primer hybridization regions that can make the amplicon shorter and therefore enhance the rate of amplification.

[0057] Various studies by the present inventors have revealed that the speed of the method and its sensitivity are enhanced by having a short amplicon, and therefore in some embodiments it may be preferable to shorten the overall length of both primers including their hybridization sequences and to position the primers with only one short gap between the first and second hybridization sequences of the target nucleic acid, for example not more than 10 or 15 nucleotide bases. In one embodiment, the first and second hybridization sequences of the target nucleic acid are separated by 0-15 or 0-6 bases, and in some embodiments, they are separated by 3-15 or 3-6 bases, for example 5, 7, or 11 bases. In further embodiments, the hybridization sequences overlap, for example by 1-2 bases.

[0058] There are several considerations in designing the sequences of oligonucleotide probes for use in this method. First, the region of the first oligonucleotide probe that hybridizes with the first single-stranded detection sequence and the region of the second oligonucleotide probe that hybridizes with the second single-stranded detection sequence are typically designed so that they do not overlap or have minimal overlap, allowing both oligonucleotide probes to bind simultaneously to at least one species in the amplification product. Also, to ensure efficient targeting to one or more species in the amplification product and to allow both oligonucleotide probes to bind to the same strand, they are typically designed to hybridize primarily to a sequence between the location of the cleavage site on one strand of the amplification product species and the location opposite the cleavage site on its reverse complementary strand. For any given primer pair, either strand can be selected for targeting by the oligonucleotide probe. Considering that the oligonucleotide probes are typically not extended by polymerase in this method, the hybridization sequences are designed based on the relevant sequences of the species in the amplification product, which determines their Tm, %GC, and experimental performance data obtained. In one embodiment, the hybridization sequence of the first and / or second oligonucleotide probe is 9-20 nucleotide bases long. In one embodiment, where the first and second hybridization sequences of the target nucleic acid are separated by 0 bases, the sequence of the hybridization region of one of the oligonucleotide probes may correspond to one of the oligonucleotide primers, and the hybridization region of the other oligonucleotide probe corresponds to the reverse complement of the other oligonucleotide primer. However, the length of the hybridization sequence can be reduced to optimize the properties of the oligonucleotide probes for a desired embodiment of the method and to avoid any inhibitory effects when all or part of step b) is performed simultaneously with step a).When the first or second oligonucleotide probe contains a recognition sequence and a cleavage site for a first or second restriction enzyme, and the oligonucleotide probe is contacted with the sample simultaneously with the implementation of step a), the cleavage site in the probe is typically blocked, for example, by including a modified internucleotide bond, such as a phosphorothioate bond, during chemical synthesis of the probe, or by introducing a mismatch to remove the recognition sequence. Besides the hybridizing region, there is considerable flexibility in the sequence of the oligonucleotide probe and any modified nucleotide bases, nucleotide bonds, or other modifications they may contain. Modified bases that can be chemically inserted into oligonucleotides to change their properties and that can be utilized in embodiments of the method, such as 2-amino-dA, 5-methyl-dC, Super T®, 2-fluoro bases, and G clamp, provide an increase in Tm, while other modified nucleotide bases, such as Iso-dC and Iso-G, can enhance binding specificity without increasing Tm. Other modifications, such as inosine or abasic sites, can reduce binding specificity. Modifications known to confer nuclease resistance include inverted dT and ddT and C3 spacers. Modifications can increase or decrease Tm, providing the potential to control hybridization events in this method embodiment. The use of modified bases in the hybridization region of an oligonucleotide probe provides the opportunity to improve the performance of the oligonucleotide probe, for example by enhancing its binding affinity without increasing the length of the hybridization region. In one embodiment, modified bases in one or both oligonucleotide probes allow them to hybridize more efficiently and therefore outperform any species in the amplification product that have complementarity with the associated single-stranded detection sequence.

[0059] In an embodiment in which one of the first and second oligonucleotide probes is blocked from extension at the 3' end and cannot be cleaved, and is contacted with the sample simultaneously with the implementation of step a), typically one oligonucleotide probe contains an additional 5' region, providing the opportunity to stabilize the predictor species as described (see FIG. 2). In one embodiment, said one oligonucleotide probe contains the exact sequence of one of the oligonucleotide primers, but contains a modification that blocks its extension by a strand-displacing DNA polymerase at the 3' end and a single phosphorothioate internucleotide bond that blocks a restriction enzyme cleavage site. Such an embodiment simplifies the assay design and ensures that no additional sequence motifs that may result in non-specific background amplification are introduced.

[0060] The first and second oligonucleotide probes for producing the detector species are preferably provided at a level such that the number of copies of the detector species produced is sufficiently above the detection limit of the means used for the easily detectable detector species. Furthermore, the efficiency of hybridization by the first and / or second oligonucleotide probes is affected by their concentration. Typically, the concentration of the oligonucleotide probes contacted with the sample simultaneously with the implementation of step a) can be similar to the concentration of the oligonucleotide primers, for example 10 to 200 nM, but this should not be considered as limiting. In one embodiment, the concentration of one or both oligonucleotide probes is provided in excess of the concentration of one or both oligonucleotide primers, whereas in another embodiment, the concentration of one or both oligonucleotide probes is provided at a lower concentration than the concentration of one or both oligonucleotide primers. When one or both oligonucleotide probes are contacted with the sample after the implementation of the amplification step a), a higher concentration allows the most efficient hybridization to be achieved as needed, and there is no need to consider any inhibition of amplification that may occur as a result of step a).

[0061] Hybridization sequence is an important feature of both oligonucleotide primers and oligonucleotide probes for the implementation of this method. Hybridization refers to sequence-specific hybridization, which is the ability of an oligonucleotide primer or probe to bind to a species in a target nucleic acid or an amplification product, resulting from hydrogen bond base pairing between complementary bases in the sequence of each nucleic acid. Typical base pairs are adenine-thymine (AT), or adenine-uracil and cytosine-guanine in the case of RNA or RNA / DNA hybrid duplexes, but various natural and non-natural analogs of nucleic acid bases are also known to have specific binding preferences. Furthermore, in the present invention, the complementary region of an oligonucleotide probe or primer does not necessarily have to contain completely natural nucleic acid bases in a sequence that has complete and exact complementarity with its hybridization sequence in a species in a target nucleic acid or an amplification product: rather, for the implementation of this method, the oligonucleotide probe / primer only needs to be capable of sequence-specific hybridization with its target hybridization sequence sufficient to form a double-stranded sequence necessary for the correct operation of the method, including cleavage by a restriction enzyme and extension by a strand-displacing DNA polymerase. Thus, such hybridization may occur even if it is not exactly complementary, but has unnatural bases or abasic sites. In one embodiment, the hybridizing region of the oligonucleotide primer or oligonucleotide probe used in the method may consist of a perfect complement to the sequence of the relevant region of the target nucleic acid, a species in the amplification product, or, if necessary, a reverse complementary sequence thereof. In other embodiments, there are one or more non-complementary base pairs. In some situations, it may be advantageous to use a mixture of oligonucleotide primers and / or probes in the method. Thus, as an example, if the target nucleic acid contains a single nucleotide polymorphism (SNP) site with two polymorphic positions, a 1:1 mixture of oligonucleotide primers and oligonucleotide probes that differ at that position (each component has complementarity to each base of the SNP) can be utilized. During the manufacture of oligonucleotides, it is a routine practice to randomize one or more bases during the synthesis process.

[0062] Those skilled in the art will appreciate that amplification processes involving polymerases can be subject to non-specific background amplification, such as that resulting from synthesis from 1 and / or interprimer binding. The methods of the present invention typically exhibit faster amplification when designed to make the amplicon as short as possible, for example by minimizing the length of the primer hybridization sequence, the gap between the first and second hybridization sequences in the target nucleic acid, and any stabilizing regions to the extent possible, while still retaining functionality at a given reaction temperature. With shorter amplicons, non-specific background may be exacerbated due to the fact that all sequences required to produce an amplified product species are provided by the oligonucleotide primers. False positive results may occur in this method if the amplicon is produced in a non-target specific manner, including both DNA synthesized from 1 or the first and second oligonucleotide primers "linked" via interprimer binding. The use of two oligonucleotide probes in this method allows the method to encompass additional features of various embodiments, minimizing the possibility of any non-target specific background signal. Such embodiments realized by using two oligonucleotide probes have a significant advantage over known methods in this respect.

[0063] One approach is to separate the first and second hybridization sequences in the target nucleic acid and provide a target-based sequence specificity check using the oligonucleotide probe of the method. Thus, in one embodiment, the first and second hybridization sequences of the target nucleic acid are separated by 3-15 or 3-6 bases, e.g., 5, 7, or 11 bases. This gap between the primers presents an optimal size gap, providing an additional specificity check of the species in the amplification product while maintaining the speed enhancement of short amplicons. Thus, in one embodiment, in step b) the first or second single-stranded detection sequence of the at least one species in the amplification product comprises a sequence of at least 3 bases corresponding to the 3-15 bases, or 3-6 bases. For example, the inventors have demonstrated the potential to distinguish between specific target-dependent amplification products and non-target-specific background amplification products, as shown in Example 4 (Figure 8).

[0064] In an alternative approach, the concentration of the first and / or second oligonucleotide primer is reduced to reduce the probability of background due to amplification from 1 and interprimer binding. To ensure that the amplification rate is maintained, an additional oligonucleotide primer that is blocked at the 3' end from extension by strand-displacing DNA polymerase can be used. In this embodiment, the unblocked first and second oligonucleotide primers are available in sufficient concentration to produce amplicons from the target nucleic acid for the first hybridization and extension event, and the subsequent amplification is continued with the blocked primer, which is preferably provided at a high concentration. Here, cleavage of the blocked primer occurs before its extension and strand displacement to remove the 3' blocking modification and allow the amplification process to continue without loss (see Figure 4). Thus, in one embodiment, in step a), the sample is further contacted with: (A) a third oligonucleotide primer, which comprises in the 5' to 3' direction the recognition sequence and cleavage site of the first restriction enzyme and one strand of a region capable of hybridizing with the first hybridization sequence in the target nucleic acid, and the extension by DNA polymerase is blocked at the 3' end; and / or (B) a fourth oligonucleotide primer, which comprises in the 5' to 3' direction the recognition sequence and cleavage site of the second restriction enzyme and one strand of a region capable of hybridizing with the reverse complement of the second hybridization sequence in the target nucleic acid, and the extension by DNA polymerase is blocked at the 3' end. In a further embodiment, the third oligonucleotide primer, if present, is provided in excess of the first oligonucleotide primer, and the fourth oligonucleotide primer, if present, is provided in excess of the second oligonucleotide primer. By significantly reducing the concentration of the first and second oligonucleotide primers and compensating for this with the presence of the third and fourth oligonucleotide primers, the greatest potential benefit is obtained in terms of removing non-target-dependent background amplification.Other than the presence of a 3' modification to block polymerase extension during oligonucleotide primer synthesis, which can be readily accomplished, for example, through the use of a 3' phosphate or C-3 modification, the same design parameters utilized for the first and second primers apply to the third and fourth primers.

[0065] Embodiments of the method of the invention that provide enhanced specificity and elimination of background amplification as described above improve the stringency of sequence confirmation, thereby allowing lower temperature reactions to be performed without loss of specificity, and / or increase multiplexing, allowing multiple reactions to be performed with simultaneous detection of multiple targets. The advantage of this stringent specificity also means that the method can tolerate a wide range of temperatures and suboptimal conditions (e.g., reagent concentrations) without loss of specificity. For example, we have performed the method with 20% increases and decreases in the concentrations of all components, and we have performed the method at ambient temperature for a significant period after performing the amplification in step a), and in each case, we have not observed any loss of specificity. Such embodiments therefore represent a significant advantage of the method of the invention over known methods, meaning that it is ideally suited for use in low-cost and / or disposable diagnostic devices.

[0066] The detection of the detector species in step c) can be achieved by any technique that differentially detects the presence of the detector species from other reagents and components present in the sample. Alternatively, the presence or level of the detector species can be inferred from the removal of one or more reaction components, such as the first or second oligonucleotide probe. From the wide variety of physicochemical techniques available for use in the detection of the detector species, a preference was given for the use of this method to techniques that are capable of generating a highly sensitive signal that is only present after hybridization of the first and second oligonucleotide probes with the relevant species in the amplification product. It will be clear to the skilled artisan that there are a variety of colorimetric or fluorometric dyes that can be easily attached to the first oligonucleotide probe, forming the basis for its visual or instrumental (e.g. absorbance or fluorescence spectroscopy) detection.

[0067] Thus, in one embodiment, the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorometric dye, or a moiety enabling binding to a colorimetric or fluorometric dye, such as biotin.

[0068] The embodiment of the method utilizing colorimetric dyes has the advantage that no fluorescent excitation and detection equipment is required, potentially allowing the presence of the target nucleic acid to be determined visually. Colorimetric detection can be achieved by directly binding a colorimetric dye, or a moiety capable of binding to a colorimetric dye, to the first oligonucleotide probe prior to its use in the method, or by specifically attaching or binding a dye or moiety to the probe fragment after cleavage. For example, the first oligonucleotide probe may contain a biotin moiety that allows its binding to a streptavidin-conjugated colorimetric dye for subsequent detection. One example of such a colorimetric dye that can be used for detection is gold nanoparticles. A variety of other intrinsically colorimetric moieties can be utilized in similar methods, of which numerous are known, such as carbon nanoparticles, silver nanoparticles, iron oxide nanoparticles, polystyrene beads, quantum dots, etc. Dyes with high extinction coefficients also offer the potential for sensitive real-time quantification in the method.

[0069] Several considerations are taken into account when selecting the appropriate dye for a given application. For example, in embodiments intended to perform visible colorimetric detection in solution, it is generally advantageous to select larger sized particles and / or particles with higher extinction coefficients for ease of detection, while embodiments incorporating lateral flow membranes intended for visible detection may benefit from the ability of smaller sized particles to diffuse more quickly along the membrane. Gold nanoparticles are available in a variety of sizes and shapes, as well as several other colorimetric moieties of interest, including polystyrene or latex-based microspheres / nanoparticles. Particles of this nature are also available in several colors. Several colors may be useful for tagging different detector species during the method and differentially detecting them, i.e., producing "multiplexed" colorimetric signals during the detection reaction.

[0070] Fluorometric detection can be achieved through the use of any dye that emits a fluorescent signal under appropriate excitation stimuli that leads to the subsequent detection of the detector species. For example, dyes for direct fluorescent detection include, but are not limited to: quantum dots, ALEXA dyes, fluorescein, ATTO dyes, rhodamine, and Texas Red. In an embodiment of the method that utilizes a fluorescent dye moiety attached to an oligonucleotide probe, it is also possible to perform detection based on fluorescence resonance energy transfer (FRET), such as using TaqMan quantitative PCR or molecular beacon-based strategies for nucleic acid detection, whereby the detector species increases or decreases after binding of the dye. In general, when using a fluorometric approach, the generation of the fluorescent signal can be recorded using several different detection devices, such as a CCD camera, a fluorescent scanner, a fluorescent-based microplate reader, or a fluorescent microscope.

[0071] In a further embodiment, the moiety that allows the detection of the first oligonucleotide probe is an enzyme that generates a detectable signal, such as a colorimetric or fluorometric signal, after contact with a substrate. Those skilled in the art will understand that several enzyme substrate systems, such as ELISA and immunohistochemical detection, are available and routinely used in the field of diagnostics. Horseradish peroxidase (HRP) is one example. Using an enzyme bound to the first oligonucleotide probe for the detection of the detector species in step c) offers several potential advantages, such as enhanced detection sensitivity and improved control of signal color development through a separate step involving the addition of substrate. Other suitable colorimetric enzymes may include: glycosyl hydrolases, peptidases or amylases, esterases (e.g., carboxyesterases), glycosidases (e.g., galactosidases), and phosphatases (e.g., alkaline phosphatases). This list should not be considered as limiting in any way.

[0072] In another approach, the presence of the detector species in step c) is detected electrically, for example by impedance change or conductivity signal change, amperometric signal change, voltammetric signal change, or potentiometric signal change.Thus, in one embodiment, the detector species is detected by the change in electrical signal.The change in electrical signal may be facilitated by a moiety that allows detection of the first oligonucleotide probe, such as a chemical group that enhances the change in electrical signal.The detection of electrical signal is very sensitive, so the detection moiety can simply be an oligonucleotide sequence, but in some embodiments, the signal is enhanced by the presence of chemical groups known to enhance electrical signals, such as metals, for example gold and carbon.

[0073] In one embodiment, the change in electrical signal due to accumulation of the detector species can be detected in the aqueous reaction during amplification, while in other embodiments, detection of the electrical signal is facilitated by localization of the detector species to specific sites, e.g., the surface of an electrochemical probe, mediated by a second oligonucleotide probe for its detection.

[0074] Other techniques routinely used to detect nucleic acids such as detector species, and which can be used for detection in this method, include mass spectrometry (e.g., MALDI or LC-TOF), emission spectroscopy or spectrometry, fluorescence spectroscopy or spectrometry, liquid chromatography, and fluorescence polarization.

[0075] In one embodiment, in step c) a colorimetric or electrochemical signal is generated using carbon or gold, preferably carbon.

[0076] In one embodiment, the detector species is detected by nucleic acid lateral flow. Nucleic acid lateral flow, in which nucleic acid is separated from other reaction components by its diffusion through a membrane, typically made of nitrocellulose, is a rapid and low-cost detection method that can combine various signal readouts, including colorimetric, fluorometric, and electrical signals. Nucleic acid lateral flow is well suited for use in the detection of detector species in this method and offers several advantages. In one embodiment, nucleic acid lateral flow detection is performed, in which a first oligonucleotide probe in the detector species is used to bind a colorimetric or fluorometric dye, and a second oligonucleotide probe in the detector species is used to localize the dye at a defined location on the lateral flow strip. In this way, rapid detection can be performed and the results can be visualized by eye or a reading device. In nucleic acid lateral flow, an antigen can be utilized as the detector moiety of the second oligonucleotide probe, and the associated antibody can be immobilized on the lateral flow strip. Alternatively, in the present method, sequence-specific detection via hybridization of the predictor or detector species on the lateral flow strip can be easily performed, providing a simplified, low-cost alternative to antibody-based assays with improved multiplexing potential. Known methods that do not utilize the two oligonucleotide probes of the present method, such as SDA, typically generate double-stranded DNA products that are not amenable to sequence-specific hybridization-based detection. In contrast, in the present method, the detector species is particularly adapted for multiplexing detection by using position-specific hybridization-based detection. Carbon or gold nanoparticles are easily accessible for nucleic acid lateral flow. Localization of the detector species results in localized carbon or gold, which appears black or red, respectively. In one embodiment, the first oligonucleotide probe contains a moiety, such as biotin, that allows binding to its colorimetric dye prior to localization on the strip by sequence-specific hybridization.

[0077] The spatial arrangement of the detector species is closely related to the technology utilized for the detection of the detector species, for example when hybridization-based binding of the detector species at a specific location is permitted. In addition to facilitating rapid and specific detection, such physical binding may enhance the use of the method in multiplex detection of multiple different target nucleic acids. In one embodiment, the second oligonucleotide probe is bound to the surface of a nucleic acid lateral flow strip or electrochemical probe, a 96-well plate, a bead, or an array surface. Thus, at least one species in the amplification product becomes localized to the physical location of the second oligonucleotide probe, which is easily detected after the formation of the detector species at such location. Alternatively, it may be advantageous to use a single-stranded oligonucleotide as the moiety that binds to the second oligonucleotide probe, which allows its binding to the solid material. In this way, the sequence of the oligonucleotide bound to the solid phase can be defined independently of the target nucleic acid sequence, enhancing the binding efficiency. Thus, in one embodiment, the moiety that allows the binding of the second oligonucleotide probe to the solid material is a single-stranded oligonucleotide. The single-stranded oligonucleotides can be designed to improve the affinity and efficiency of hybridization, enhancing the performance of the method. For example, in one embodiment of the method, rather than directly attaching the second oligonucleotide probe to the lateral flow strip, a separate oligonucleotide is employed that has a sequence optimized for on-strip hybridization that allows efficient hybridization with the single-stranded oligonucleotide moiety present in the second oligonucleotide probe.

[0078] In various studies, the present inventors have significantly improved the performance of nucleic acid lateral flow methods using single-stranded oligonucleotides as the binding moiety of the second oligonucleotide probe, and enhanced the hybridization sequence on the strip. For example, GC-rich sequences can be used for on-strip hybridization, or longer sequences with higher Tm can be used to complement the length of the second oligonucleotide probe. Alternatively, the single-stranded oligonucleotide portion can contain one or more modified bases or internucleotide bonds to enhance its affinity, such as PNA, LNA, or G-clamp. The present inventors have recognized that when a repeat sequence motif is used in the single-stranded oligonucleotide portion, an unexpected enhancement in hybridization efficiency is observed that is not predicted by its predicted Tm. Thus, in one embodiment, the sequence of the single-stranded oligonucleotide portion contains three or more repeat copies of a 2-4 base DNA sequence motif. For example, in various studies using such sequence motifs, the present inventors have observed that the detection sensitivity by nucleic acid lateral flow is greatly enhanced, often with a signal enhancement of 100 times or more.

[0079] Thus, in one embodiment in which the presence of the detector species is detected by nucleic acid lateral flow, said nucleic acid lateral flow utilises one or more nucleic acids capable of sequence-specific hybridisation with a moiety enabling binding of said second oligonucleotide probe to a solid material.

[0080] Further advantages are conferred by separating the target nucleic acid sequence from the solid material for binding or from the detection means. This is possible by using single-stranded oligonucleotides as detection moieties in the first oligonucleotide probe and / or as binding moieties with the second oligonucleotide probe. In this way, the relevant solid material for binding or the device comprising said solid material, e.g., a nucleic acid lateral flow strip, and / or the detection means can be optimized and defined without taking into account the sequence of the target nucleic acid to be detected. Such a "universal" detection device can be used without the need to change for each application and target. For example, a nucleic acid lateral flow strip printed with lines corresponding to a matching set of oligonucleotide sequences capable of efficient on-strip hybridization and not defining unintended crosstalk, optimized and efficiently manufactured independently of the development of the oligonucleotide primers and probes of the method for the detection of multiple target nucleic acid sequences.

[0081] In some embodiments, the detection can be performed quantitatively. Thus, the level of single-stranded target nucleic acid in the sample can be quantified in step c). Quantification can be achieved by measuring the detector species, for example colorimetrically, fluorometrically, or electrically, at multiple points in the time series of the reaction, rather than at a single end point. Alternative strategies for quantification include serial dilution of the sample, similar to droplet digital PCR. In further embodiments, the level of single-stranded target nucleic acid in the sample can be determined semi-quantitatively. For example, the intensity of the colorimetric signal on a nucleic acid lateral flow strip corresponds to the approximate level of single-stranded target nucleic acid in the sample. Alternatively, an inhibitor can be used, whereby the copy number of the single-stranded nucleic acid target must exceed a certain defined copy number in order to overcome the inhibitor and produce a detectable copy number of the detector species.

[0082] In the method of the present invention, the second oligonucleotide probe is bound to a solid material or a moiety that allows binding to the solid material. In an embodiment, one or more other oligonucleotide primers and probes can also be optionally bound to a solid material or a moiety that allows binding to the solid material. It will be clear to those skilled in the art that the binding of oligonucleotides to solid materials can be achieved in a variety of different ways. For example, several different solid materials are available that have a sufficient density of functional groups or can be bound to or functionalized with appropriately modified oligonucleotide probes to be useful for the purpose of binding or reacting with them. Furthermore, such solid materials are available in a wide variety of shapes, sizes, and forms, including beads, resins, surface-coated plates, slides, and capillaries. Examples of such solid materials used for covalent attachment of oligonucleotides include, but are not limited to, glass slides, glass beads, ferrite core polymer coated magnetic microbeads, silica microparticles or magnetic silica microparticles, silica-based capillary microtubes, 3D reactive polymer slides, microplate wells, polystyrene beads, poly(lactic acid) (PLA) particles, poly(methyl methacrylate) (PMMA) microparticles, controlled pore glass resins, graphene oxide surfaces, and functionalized agarose or polyacrylamide surfaces. Polymers such as polyacrylamide have the added advantage that functionalized oligonucleotides can be covalently attached during the polymerization reaction between the monomers (e.g., acrylamide monomers) used to generate the polymer. Functionalized oligonucleotides are included in the polymerization reaction to produce solid polymers containing covalently attached oligonucleotides. Such polymerization provides a highly efficient means of attaching oligonucleotides to solid materials with control over the size, shape, and morphology of the resulting oligonucleotide-bound solid material.

[0083] Typically, to attach an oligonucleotide probe to any such solid-state material, an oligonucleotide is synthesized with a functional group at the 3' or 5' end, although a functional group can also be added at almost any base position during the oligonucleotide production process. A specific reaction can then be carried out between the functional group in the oligonucleotide and the functional group on the relevant solid-state material to form a stable covalent bond, resulting in an oligonucleotide attached to the solid-state material. Typically, such an oligonucleotide is attached to the solid-state material at the 5' or 3' end. As an example, two commonly used and reliable attachment chemistries utilize thiol (SH) or amine (NH3) groups and functional groups in the oligonucleotide. Thiol groups can react with maleimide moieties on the solid support to form thioester bonds, while amines can react with maleimide moieties on the solid support to form thioester bonds. can react with succinimidyl ester (NHS ester) modified carboxylic acids to form amide bonds. Several other chemistries can also be used. In addition to chemical conjugation of oligonucleotide probes to solid-state materials, it is possible and potentially advantageous to synthesize oligonucleotide probes directly on solid-state materials for use in carrying out the method.

[0084] In other embodiments, the second oligonucleotide probe is bound to a moiety that allows it to bind to the solid material.One strategy utilizes the method of affinity binding, whereby a moiety that allows specific binding can be bound to the oligonucleotide probe to promote binding to its associated affinity ligand.This can be done, for example, by using affinity tags such as antibody antigen binding or polyhistidine tags, or by using nucleic acid-based hybridization to bind complementary nucleic acid to solid material, for example nitrocellulose nucleic acid lateral flow strips.An exemplary such moiety is biotin.Biotin can bind with high affinity to streptavidin or avidin that is itself bound to beads or another solid surface.

[0085] The presence of two or more different target nucleic acids of defined sequence can be detected in the same sample. In one embodiment of the method, a separate sequence of steps a), b), and c) is performed using different oligonucleotide primers and oligonucleotide probes for each of the two or more target nucleic acids. Here, the separate steps can be performed simultaneously. For example, in one embodiment, one set of oligonucleotide primers and oligonucleotide probes is used to detect one target nucleic acid in a sample, and another set of oligonucleotide primers and oligonucleotide probes is used to detect another target nucleic acid in the same sample. The detection of detector species produced from two or more different sets of primers / probes can each be coupled to a specific signal that allows multiplex detection, such as a different colorimetric or fluorometric dye or enzyme. Alternatively, multiplex detection can be achieved by binding a second oligonucleotide probe to a solid material directly or indirectly through a moiety that allows binding to the solid material. Such an approach does not rely on different detection means, but instead utilizes the physical separation of detector species produced by different sequences of steps a), b), and c). Thus, for example, a single dye can be used on a nucleic acid lateral flow strip to detect multiple different target nucleic acids, with each different detector species produced localizing to a specific printed line on the lateral flow strip and forming the basis for differentiated detection by direct or indirect sequence-based hybridization with a second oligonucleotide probe. Alternatively, an electronic detection array can be used, with multiple different second oligonucleotide probes attached to specific regions of the array, thus creating a multiplex reaction and simultaneously producing multiple different detector species, each detector species localized by hybridization to discrete regions of the array allowing multiplex detection.

[0086] The above detection processes, such as nucleic acid lateral flow and electrical detection, and their ability to easily detect multiple different target nucleic acids in the same sample, are achieved by the method essentially requiring two oligonucleotide probes, thus effectively demonstrating the advantages of the method of the present invention over known methods.

[0087] The present invention has broad utility for a variety of fields and applications requiring detection of a target nucleic acid of a defined sequence in a sample. The present invention provides a means for rapid, inexpensive, and convenient determination of the presence of a target nucleic acid sequence in a sample. Through a non-limiting list of applications, the inventors envision the present invention being valuable in fields such as diagnostics, forensics, agriculture, animal health, environment, defense, human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics or pharmacokinetics, as well as microbiology, clinical and biomedical research. Preferably, the sample is a biological sample, such as a human sample. The sample may be a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample.

[0088] Detection of target nucleic acids may be used for the diagnosis, prognosis, or monitoring of a disease or condition, such as, but not limited to, an infectious disease, including, but not limited to, HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox, or a cancer, including, but not limited to, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma, or in the fields of human genetic testing, prenatal testing, blood contamination screening, pharmacogenetics, or pharmacokinetics.

[0089] The present invention may be used with a wide variety of samples such as, for example: nasal swabs or aspirates, nasopharyngeal swabs or aspirates, throat swabs or aspirates, buccal swabs or aspirates, blood or blood derived samples, urine or urine derived samples, sputum or sputum derived samples, faeces or faeces derived samples, cerebrospinal fluid (CSF) or CSF derived samples, and gastric juice or gastric juice derived samples, any form of tissue biopsy or body fluid derived human or animal sample. The inventors have also performed this method on a wide range of samples including the following clinical specimens: nasal swabs of VTM, nasopharyngeal swabs of VTM, thin prep medium, throat swabs in liquid Amies, HSV sore swabs in M4 medium, synovial fluid, sputum treated with 2M NaOH / isopropanol followed by DNA capture beads, rectal swabs in TE, fecal samples homogenized and treated with DNA capture beads, CSF, APTIMA swabs, amniotic fluid, oral swabs in liquid Amies, urine, VRE swabs in TE, pleural fluid, whole blood, K2EDTA plasma, L. heparin plasma, and at least 10-20% of blood serum. These experiments demonstrate the remarkable versatility of this method for various clinical applications and the lack of inhibition observed in the relevant samples. This is in stark contrast to other methods that are inhibited by inhibitors found in biological samples, such as heparin and phytic acid, which inhibit PCR, and thus demonstrates the potential of using this method in low-cost or disposable devices without requiring any complex sample preparation procedures.

[0090] The target nucleic acid may be, inter alia, (a) from a virus or viral nucleic acid material, (b) from a bacteria or bacterial nucleic acid material, (c) circulating cell-free DNA released from a cancer cell, (d) circulating cell-free DNA released from a fetal cell, or (e) from a microRNA or microRNA.

[0091] The single-stranded target nucleic acid of the method may be naturally occurring or non-naturally occurring. The target nucleic acid may be generated in situ or produced from natural nucleic acid before carrying out the method. The single-stranded target nucleic acid of the method may be prepared by one or more additional steps carried out before or simultaneously with step a), which may include one or more enzymes, such as polymerases and restriction enzymes. Such generation of the target nucleic acid of the method has several potential advantages, such as enabling even higher multiplex assays and / or overcoming background from 1. For example, highly specific conversion of nucleic acid material in a biological sample can be carried out without amplification before the amplification in step a). The sample can be, for example, processed, purified, subjected to buffer exchange, subjected to exome capture, partially removed contaminants, and / or converted to single-stranded target nucleic acid for the method involving one or more modified dNTPs. If the "real" target nucleic acid to be detected in the sample is converted into a "surrogate" target nucleic acid for the implementation of the method by a reliable conversion (which may be <1:1, 1:1 or 1:>1, i.e. may have some amplification elements), the detection of the "surrogate" target nucleic acid will allow the "real" nucleic acid to be detected and / or quantified. Furthermore, the production of a surrogate target from a natural target can thus be used to specifically generate a target nucleic acid of the method having any desired sequence. In one embodiment where the single-stranded target nucleic acid is obtained from double-stranded DNA after dissociation of the two strands, for example by strand invasion, there are two complementary single-stranded nucleic acid targets, which can be amplified and detected in a reciprocal process by the same oligonucleotide primers and probes. If the target is the genome of a -ve strand single-stranded RNA virus, there may also be +ve strand transcripts in the sample, and either or both strands can be amplified and detected as single-stranded target nucleic acid in this method using the same oligonucleotide primers and probes.

[0092] It is also envisioned that the present invention has the potential to be useful in screening samples for cell-free DNA and epigenetic modifications, such as CpG methylation of DNA sequences. Such epigenetic modifications of specific cancer-related target genes can serve as useful biomarkers in many diseases and disease states. Given the increasing appreciation of the importance of epigenetic modifications in human diseases, the present invention has the potential to be used to specifically evaluate epigenetic modifications of specific target nucleic acid biomarkers based on the differential activity of strand-displacing DNA polymerases and / or restriction enzymes. Thus, in one embodiment, the target nucleic acid comprises an epigenetic modification site, such as methylation. Alternatively, the "true" nucleic acid used to produce a "surrogate" target nucleic acid for carrying out the above method comprises an epigenetic modification site.

[0093] A further aspect of the present invention relates to a kit for use in the detection of a nucleic acid of a defined sequence in a sample. a) a first oligonucleotide primer and a second oligonucleotide primer, the first primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to a first hybridization sequence in a single-stranded target nucleic acid of defined sequence, and the second primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; b) a first restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the first primer and cleaving the cleavage site, and a second restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the second primer and cleaving the cleavage site; c) strand-displacing DNA polymerase; d) dNTPs; e) one or more modified dNTPs; f) a first oligonucleotide probe that is capable of hybridizing to and permitting detection of at least one species of a first single-stranded detection sequence in an amplification product produced in the presence of the target nucleic acid; and g) a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of said first single-stranded detection sequence of said at least one species in the amplification product and attached to a solid material or a moiety that allows binding to said solid material.

[0094] In one embodiment, one of the first and second oligonucleotide probes of the kit is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, e.g. due to the presence of one or more sequence mismatches and / or one or more modifications, e.g. phosphorothioate linkages.

[0095] In one embodiment, one of the first and second oligonucleotide probes of the kit has 5 or more bases of complementarity with the hybridizing region of the first or second primer or the reverse complement of the hybridizing region of the first or second primer.

[0096] In another embodiment, the first oligonucleotide probe of the kit has some complementarity, e.g., 5 or more bases of complementarity, with the hybridizing region of one of the first and second oligonucleotide primers, and / or the second oligonucleotide probe of the kit has some complementarity, e.g., 5 or more bases of complementarity, with the reverse complement of the hybridizing region of the other of the first and second oligonucleotide primers.

[0097] In further embodiments, as described above, the first and / or second oligonucleotide probe may have some complementarity or reverse complementarity to the gap between the first and second hybridization sequences of the target nucleic acid.

[0098] The kit may also include a reverse transcriptase.

[0099] The kit may further comprise means for detecting the presence of a detector species produced in the presence of said target nucleic acid, for example the kit may further comprise a nucleic acid lateral flow strip, an electrochemical probe, a 96 well plate, beads or array surface, and / or a colorimetric or fluorometric dye, and / or a device for detecting a change in an electrical signal, and / or carbon or gold.

[0100] In various embodiments, the target nucleic acid and kit components contained in the kit, such as the first oligonucleotide primer, and / or the second oligonucleotide primer, and / or the first restriction enzyme, and / or the second restriction enzyme, and / or the DNA polymerase, and / or the dNTPs, and / or the one or more modified dNTPs, and / or the first oligonucleotide probe, and / or the second oligonucleotide probe, and / or the first or second single-stranded detection sequence in at least one species in the amplification product, are as defined herein for the method of the invention. For example, the kit may include any combination of the features of the components described herein, including, but not limited to, the following: one of the first and second oligonucleotide probes is blocked at its 3' end from extension by a DNA polymerase and cannot be cleaved by said first or second restriction enzyme, optionally due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate linkages; said first restriction enzyme and second restriction enzyme are the same restriction enzyme; said one or more modified dNTPs are α-thiol modified dNTPs; the detectable moiety of the first oligonucleotide probe is a colorimetric or fluorometric dye or a moiety that can be bound to a colorimetric or fluorometric dye, such as biotin; the portion of the oligonucleotide probe enabling binding to the solid material is optionally a single-stranded oligonucleotide comprising 3 or more repeated copies of a DNA sequence motif of 2-4 bases; said first and second oligonucleotide primers comprise a stabilizing sequence upstream of said restriction enzyme recognition sequence and said cleavage site, e.g. at the 5' end, and e.g. 5 or 6 bases long; the hybridizing region of the first and / or second oligonucleotide primer is 6-30 bases long, e.g. 9-16 bases long; and the first and second hybridization sequences of the target nucleic acid are separated by 0-15 or 0-6 bases, in certain embodiments they are separated by 3-15 or 3-6 bases, e.g. 5, 7, or 11 bases, or they overlap by e.g. 1-2 bases.

[0101] The kit may comprise means for detecting the presence of said detector species produced in the presence of said target nucleic acid, such as a nucleic acid lateral flow strip. In a further embodiment, the kit further comprises a third and / or a fourth oligonucleotide primer as defined herein.

[0102] The kit may also include reagents such as reaction buffers, salts, eg, divalent metal ions, additives, and excipients.

[0103] Kits according to the invention may be provided together with instructions for the practice of the methods according to the invention.

[0104] The present invention also provides the use of a kit of the present invention for detecting a single-stranded target nucleic acid of defined sequence in a sample.

[0105] It is to be understood that all optional and / or preferred embodiments of the invention described herein in relation to the method of the invention also apply in relation to the kit of the invention and its uses, and vice versa.

[0106] As mentioned above, the methods and kits of the invention are ideally suited for use in devices such as disposable diagnostic devices. Thus, the invention also provides kits comprising a device as described above, in particular a kit comprising a means for detecting the presence of a detector species produced in the presence of said target nucleic acid, such as a nucleic acid lateral flow strip. The device may be a powered device, such as an electrically powered device, and the device may include heating means and may be a self-contained device, i.e. a device that does not require auxiliary testing equipment.

[0107] The method of the invention can also be used independently of the detection step c) for amplifying a nucleic acid signal derived from a target nucleic acid of defined sequence, such a method can be used, for example, where the amplified signal is to be stored and / or transported for detecting the target nucleic acid at a future time and / or at an alternative location, as required. The amplified signal comprises a predictor or detector species produced during the implementation of the method. Thus, in a further embodiment, the invention provides a method for amplifying a nucleic acid signal derived from a target nucleic acid of defined sequence in a sample, said method comprising step a) and all or part, for example part i. or ii. of step b) of the method of the invention.

[0108] The present invention also provides the use of a kit according to the invention for amplifying a nucleic acid signal derived from a target nucleic acid of defined sequence as defined above.

[0109] It is to be understood that all optional and / or preferred embodiments of the invention described herein in relation to the method of the invention for detecting the presence of a target nucleic acid of defined sequence in a sample apply in relation to a method for amplifying a nucleic acid signal derived from a target nucleic acid of defined sequence.

[0110] The following examples serve to further demonstrate various aspects and embodiments of the methods described herein and should not be construed as limiting in any manner. EXAMPLES

[0111] (Example) Materials and Methods The following materials and methods are used in the examples below unless otherwise indicated. Oligonucleotides: Unless otherwise indicated, custom oligonucleotides were produced by Integrated DNA Technologies using the phosphoramidite method.

[0112] Nucleic acid lateral flow: Carbon nanoparticles were conjugated with various biotin-binding proteins, such as streptavidin, via non-covalent adsorption. Typically, colloidal carbon suspensions were prepared in borate buffer, followed by sonication using a probe sonicator. The carbon was then adsorbed to the biotin-binding proteins by incubation at room temperature. The carbon was either used directly in the reaction mixture or applied to a glass fiber conjugate pad. The lateral flow strips were constructed by combining a conjugate pad containing lyophilized sugars and additives used to improve visual appearance with a sample pad, a nitrocellulose membrane, and an adsorbent pad, according to the manufacturer's (Merck Millipore) guidelines. Prior to its use in the lateral flow strip, relevant oligonucleotides containing the reverse complement of the sequence to be detected in this method were printed at defined locations on the nitrocellulose membrane and bound to the membrane via UV crosslinking.

[0113] Example 1 (Implementation of the method in which a second oligonucleotide probe is bound to a solid material, a nitrocellulose lateral flow strip) This example illustrates the implementation of a method in which a second oligonucleotide probe is bound to a solid material, a nitrocellulose lateral flow strip, and the first oligonucleotide probe is not contacted with the sample simultaneously with the implementation of the amplification step a).

[0114] A first oligonucleotide primer was designed with a total length of 24 bases, including in the 5' to 3' direction: a 7-base stabilizing region; a 5-base non-nicking restriction enzyme recognition sequence; and a 12-base hybridization region that includes the reverse complement of the first hybridization sequence in the target nucleic acid. A second oligonucleotide primer was designed with the same stabilizing region and restriction enzyme recognition sequence, but with a 12-base hybridization region that can hybridize with the reverse complement of the second hybridization sequence in the target nucleic acid. In this example, the first and second restriction enzymes are the same restriction enzyme. The restriction enzyme is an asymmetric double-stranded cut restriction enzyme with a top strand cut site downstream of the 5-base recognition sequence. The first and second hybridization sequences in the target nucleic acid are separated by one base.

[0115] An oligonucleotide primer is designed using the target nucleic acid so that the nucleotide base downstream of the cleavage site on the reverse complement of the primer is adenosine, such that α-thiol dATP is utilized as the modified dNTP in this method. Phosphorothioate modifications are inserted by a strand displacing polymerase to prevent cleavage of the reverse complement.

[0116] A first oligonucleotide probe was designed with a total length of 20 bases, including in the 5' to 3' direction: a 12 base region complementary to at least one species in the amplification product; a 6 base intermediate spacer region; and a 3' biotin modification added during synthesis, where the biotin modification allows the first oligonucleotide probe to be conjugated to a colorimetric dye, carbon nanoparticles. Carbon adsorbed with biotin-binding protein was prepared and saturated with the first oligonucleotide probe. A second oligonucleotide probe was designed with a total length of 49 bases, including in the 5' to 3' direction: an intermediate spacer including 10x thymidine bases; and a 3x repeat sequence of a 13 base region capable of hybridizing to a second single-stranded detection sequence downstream of the first single-stranded detection sequence in the at least one species in the amplification product. Approximately 30 pmol of the second oligonucleotide probe was printed onto a nucleic acid flow strip.

[0117] Reactions were prepared containing 1.6 pmol of first primer; 0.1 pmol of second primer; 250 μM 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) SP-isomer (Sp-dATP-α-S) from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand-displacing DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at various levels (++=1 amol, +=10 zmol, NTC=no target control) in a total reaction volume of 10 μl in the appropriate reaction buffer. Reactions were incubated at 45°C for 7 or 10 min. Subsequently, 6.5 μl of termination reaction mix was added at 0.056 mg ml -1 After addition of 60 μl of lateral flow running buffer containing conjugated carbon, a second oligonucleotide probe was loaded that was attached to the nucleic acid lateral flow strip at the line printed on the strip.

[0118] Figure 5 shows a photograph of the lateral flow strip obtained in the practice of this example. The arrow indicates the position where the second oligonucleotide probe was printed onto the nitrocellulose strip, thus resulting in the appearance of a positive signal. A distinct black line, corresponding to the presence of a carbon signal, was observed only in the presence of target nucleic acid at both target levels and at both time points, demonstrating the rapid and sensitive detection of target nucleic acid sequences by the method of the present invention.

[0119] Example 2 (Implementation of the method, in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with a sample in step a)) This example demonstrates the performance of an embodiment of the method, in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with the performance of step a). In such an embodiment, the inventors did not observe any significant inhibition of the amplification rate, demonstrating that the predictor species was accumulated in real time without interrupting the optimal cyclic amplification process. Not only did the inventors observe no inhibitory effect on the amplification process in the embodiment, but the inventors also observed an unexpected enhancement in the generated signal corresponding to at least a 100-fold increase in the amount of detector species.

[0120] (Example 2.1) A variation of the assay used in Example 1 was designed utilizing an embodiment of the method in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with performing step a). The same oligonucleotide primers, restriction enzymes, dNTPs, modified dNTPs, and polymerase as utilized in Example 1 were used, but an alternative first oligonucleotide probe of full length 21 bases was designed that contains, in the 5' to 3' direction: a 5' biotin modification; an 8 base middle region; a 13 base region capable of hybridizing to at least one species in the amplification product; and a 3' phosphate modification (the biotin modification allows the first oligonucleotide probe to be conjugated to a colorimetric dye, carbon nanoparticles. The phosphate modification prevents its extension by a strand-displacing DNA polymerase). Carbon adsorbed with biotin-binding protein was prepared and saturated with the first oligonucleotide probe.

[0121] An alternative second oligonucleotide probe was designed with a total length of 51 bases, including in the 5' to 3' direction: a 14 base region capable of hybridizing to a second single-stranded detection sequence upstream of the first single-stranded detection sequence in the at least one species in the amplification product; a 6 base intermediate spacer sequence; repeats of the 14 base hybridizing region; a second 6 base intermediate spacer sequence; and a 10x thymidine-based spacer. Approximately 30 pmol of the second oligonucleotide probe was printed onto a nucleic acid flow strip.

[0122] Reactions were prepared containing 0.8 pmol of first primer; 0.8 pmol of second primer; 0.6 pmol of first oligonucleotide probe; 300 μM Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand-displacing DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at various levels (++=1 amol, +=10 zmol, NTC=no target control) in a total reaction volume of 10 μl in the appropriate reaction buffer. Reactions were incubated at 45°C for 6 min. 5 μl of termination reaction mix was then added at 0.03 mg ml -1 The nucleic acid was added to 60 μl of lateral flow running buffer containing 100 μl of conjugated carbon and then loaded onto the nucleic acid lateral flow strip. A control reaction was performed to demonstrate that no detector species was produced in the absence of the first oligonucleotide probe in the reaction. An equivalent level (0.6 pmol) of probe was added to the control after step a) to control for any unintended effects of the presence of the probe during detection on the lateral flow strip.

[0123] FIG. 6A presents a photograph of the nucleic acid lateral flow strip after its development. When the first oligonucleotide probe was provided during the reaction, a clear signal corresponding to the deposition of carbon nanoparticles was observed at both target levels. As expected, when the first oligonucleotide was not provided during the reaction, no signal was detected at either target level. This example clearly demonstrates the potential to significantly enhance the production of detector species in an embodiment of the method in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with the implementation of step a). It is noteworthy that, in contrast to example 1, equal concentrations of the first and second oligonucleotide primers are provided, allowing for more rapid amplification.

[0124] Example 2.2 Next, a separate assay was designed to demonstrate the versatility of the above embodiment of the method with a completely different target nucleic acid. Oligonucleotide primers and oligonucleotide probes were designed for a related target nucleic acid, a single-stranded DNA target, similar to those described in Examples 1 and 2.1.

[0125] Reactions were prepared containing 0.8 pmol of first primer; 0.4 pmol of second primer; 0.6 pmol of first oligonucleotide probe; 300 μM Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand-displacing DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at various levels (+=1 amol, NTC=no target control) in a total reaction volume of 10 μl in the appropriate reaction buffer. Reactions were incubated at 45°C for 6 min. 5 μl of termination reaction mix was then added at 0.08 mg ml -1 The nucleic acid was added to 60 μl of lateral flow running buffer containing conjugated carbon and then loaded onto the nucleic acid lateral flow strip. A control reaction was performed containing a shortened variant of the first oligonucleotide probe that was also contacted with the sample at the same time as step a) was performed.

[0126] FIG. 6B presents a photograph of the nucleic acid lateral flow strip after its development. In the presence of target nucleic acid, a clear positive signal was visible, and no signal was visible in the no target control. This demonstrated the proper design of the assay operation and the robust potential of the embodiment of the method in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with the implementation of step a). As expected, only a minimal signal was observed in the control assay utilizing a truncated form of the first oligonucleotide probe, demonstrating that proper hybridization of the first oligonucleotide probe is required simultaneously with the implementation of the amplification in step a) to efficiently produce detector species.

[0127] Example 3 (Implementation of the method, in which the presence of two or more different target nucleic acids of defined sequence is detected in the same sample) This example demonstrates the potential of the method to detect two or more different target nucleic acids of defined sequence in a sample. The use of two oligonucleotide probes in addition to primers in this method provides an essential approach to detect amplification products in a manner ideally suited for the detection of two or more different target nucleic acids in the same sample. In this example, it is demonstrated that alternative detector species can be differentially detected based on the sequence-specific hybridization of the second oligonucleotide probe.

[0128] First, to demonstrate that this method can be utilized to detect two or more different target nucleic acids, we developed a compatible set of oligonucleotide primers and probes for detecting two different targets (A and B). In each case, a first oligonucleotide probe was designed that contains the following features in the 5' to 3' direction: a 5' biotin modification, a 7-base stabilizing region, a 5-base restriction endonuclease recognition site, an 11-13 base region complementary to the 3' end of target A or B containing a phosphorothioate bond at the restriction enzyme cleavage site, and a 3' phosphate modification. A second oligonucleotide probe was designed that contains in the 5' to 3' direction: a 12-14 base region complementary to the 5' end of target A or B, a 5× thymidine base intermediate spacer, and a 12-base single-stranded oligonucleotide portion that allows binding of the second oligonucleotide probe to the solid material. The sequence of the single-stranded oligonucleotide binding portion of each target was designed with a different sequence to allow binding to different locations on the lateral flow strip of each detector type. A lateral flow strip of nucleic acid was prepared containing, at separate locations, 30 pmol of discrete spots of oligonucleotides containing reverse complementary sequences to each single-stranded oligonucleotide detection moiety.

[0129] 0.032 mg / ml -1 Reactions were set up containing 0.5 pmol of first oligonucleotide probe for target A and target B; 0.5 pmol of second oligonucleotide primer for targets A and B in 65 μl of appropriate buffer containing carbon adsorbed to biotin-binding protein. Separate reactions were run with different levels of each target (+=0.1 pmol, ++=1 pmol) and both targets were added together. A no target control (NTC) was also performed.

[0130] Figure 7A shows a photograph of the lateral flow strips obtained in this experiment. Distinct dark spots corresponding to the deposition of carbon containing detector species were observed for both assays at both target levels. Furthermore, signals corresponding to both targets A and B were observed when both reactions were performed simultaneously. No background signal or crosstalk between the different assays was observed.

[0131] To demonstrate the robustness of the method, further experiments were continued to develop three separate assays to demonstrate the potential of the method for detection of three different target nucleic acids of defined sequences in a sample. The same methodology as above was used. Figure 7B shows a photograph of the resulting lateral flow strip. Targets P1, P2, and P3 were added individually and in various combinations as indicated. The reverse complement to the single-stranded oligonucleotide detection portion of the second oligonucleotide probe was printed in a separate line on the nucleic acid lateral flow strip. The black signal indicates the deposition of carbon-bound detector species localized in the predicted position for rapid and sensitive detection in all cases, without unintended crosstalk between assays or any background signal. A comparable experiment involving four separate assays demonstrates the potential of the method for detection of four different target nucleic acids of defined sequences (P1, P2, P3, and P4) in a sample. The results are shown in Figure 7C. In this four-target experiment, P4 was present in all reactions as a positive control, and the other targets were added separately in separate reactions. The photograph of the lateral flow strip shown shows a clear black band at the expected location corresponding to the presence of the relevant detector species bound to the carbon. Such a multiplex assay demonstrates the potential of the method to be used for diagnostic testing of diseases caused by several different pathogens, where detecting the presence of the detector species of the control assay indicates the successful implementation of the method, and visualization of one or more other detector species on the lateral flow strip indicates the presence of the relevant causative pathogen in the appropriate clinical specimen. Although observing co-infections where two or more pathogens are present in the same specimen is rare in such diagnostic applications, e.g. in the field of infectious diseases, the method of the invention has a high degree of versatility for any combination of targets of the multiplex reaction to be detected. In Figure 7D, the results of an experiment in which different combinations of four targets (P1, P2, P3, and P4) are added are shown. The ability to detect each target separately and to detect the other three targets when each target is omitted without generating non-specific background demonstrates the remarkable specificity of detection of the method of the invention.

[0132] In the above and various other experiments, the inventors have also performed multiplex assays to detect 3-5 targets at very low target concentrations, e.g., 1 zmol (600 copies) or 17 ymol (10 copies). In this example, the inventors clearly demonstrated the potential of the method to detect the presence of two or more different target nucleic acids of defined sequences in a sample, and its potential for rapid, low-cost signal detection, e.g., by nucleic acid lateral flow. It is an exceptionally advantageous feature of the method of the present invention that two or more different target nucleic acids of defined sequences can be easily detected in the same sample. For each additional target to be detected, an additional set of oligonucleotide primers is required. This presents a significant difficulty in detecting the presence of two or more different target nucleic acids in prior art methods that do not involve temperature cycling, since the additional primers increase the tendency to form non-specific amplification products. In the method of the present invention, this challenge is overcome by the enhanced specificity resulting from, e.g., the use of modified bases, the selection of improved enzymes, and the formation of detector species using oligonucleotide probes that exploit additional sequence-specific hybridization events.

[0133] Example 4 (Implementation of the method wherein the first and second hybridization sequences in the target nucleic acid are separated by 5 bases) This example demonstrates the implementation of the method, in which the first and second hybridization sequences in the target nucleic acid are separated by 5 bases. The ability to use target-derived sequences that are not present in the oligonucleotide primers and are only produced in the amplification product in a target-dependent manner when the two oligonucleotide primers are designed to have a gap between the first and second hybridization sequences provides the potential for enhanced specificity in the embodiment of the method, which can overcome any background signal resulting from synthesis from one or interprimer binding. In the embodiment, the sequence-specific hybridization of the first or second oligonucleotide probe is designed with a gap between the two hybridization regions so that a detector species is produced only if the amplification product contains the appropriate target-derived sequence.

[0134] In this example, we designed various assays to demonstrate the hybridization of a second oligonucleotide probe to a variety of different amplification products that differ only in the sequence of the gap between the first and second hybridization sequences in the target nucleic acid. The second oligonucleotide probe was designed to contain an 11-base hybridization region at its 5' end for at least one species in the amplification product. The region is composed of a 7-base sequence that is the reverse complementary sequence of the first oligonucleotide primer and a 5-base sequence that is the reverse complementary sequence to an additional target-derived sequence in the amplification product obtained from the gap between the two primers. The second oligonucleotide probe also contains, in the 5' to 3' direction, a 5x thymidine base intermediate spacer and a 12-base single-stranded oligonucleotide portion for binding to the solid material. A nitrocellulose nucleic acid flow strip was prepared on which 30 pmol of oligonucleotide containing the reverse complementary sequence of the portion was printed. The first oligonucleotide probe was designed to be identical in sequence to the second oligonucleotide primer, but to contain a 5' biotin modification, a 3' phosphate modification, and a phosphorothioate internucleotide linkage at the restriction enzyme cleavage site.

[0135] Four different artificial target nucleic acid sequences (T1, T2, T3, and T4) were designed, each with the exact sequence corresponding to the first and second hybridization sequences, but with five base differences between the first and second hybridization sequences: T1 contains bases suitable for detection with perfect complementarity to the 11-base hybridization region of the second oligonucleotide probe; T2 contains four mismatches out of the five bases of the gap; T3 is designed such that four bases out of the five bases of the gap are removed, thus making the amplification product species four bases shorter; and T4 contains two mismatches out of the five bases of the gap.

[0136] Reactions were assembled containing 3.6 pmol of the first oligonucleotide primer; 1.8 pmol of the second oligonucleotide primer; 2.4 pmol of the first oligonucleotide probe; 300 μM Sp-dATP-α-S, 60 μM dTTP, dCTP, dGTP; 12 U of restriction enzyme; and 12 U of Bacillus strand-displacing DNA polymerase in a total reaction volume of 60 μl in the appropriate reaction buffer. After adding 1 amol target (T1, T2, T3, or T4) to each reaction, the reaction was incubated at 45° C. for 6.5 minutes before running 53.5 μl of the 60 μl reaction on a lateral flow strip. Prior to application of the reaction to the lateral flow strip, 1.5 pmol of the second oligonucleotide probe and 2 μg of biotin-binding protein adsorbed carbon were deposited on the conjugate pad and allowed to dry for 5 minutes.

[0137] Figure 8 shows a photograph of the nucleic acid lateral flow strips obtained in this experiment. The strip obtained with target T1 showed a clear black line corresponding to the carbon bound to the detector species bound to the nitrocellulose solid material, demonstrating that the assay developed in this example, including the oligonucleotide primers and probes, worked properly and had the potential for rapid and sensitive detection. The reactions performed with targets T2 and T3 did not show any carbon corresponding to a positive signal, demonstrating that both the four mismatches and the removal of four bases caused the second oligonucleotide to lose its ability to hybridize efficiently with the predictor species produced in the reaction. A very faint signal was observed on the strip produced using T4, indicating that the presence of as few as two mismatched bases resulted in a significant decrease in the ability of the second oligonucleotide probe to successfully hybridize with the predictor species to produce a detector species that could bind to the line on the strip. Polyacrylamide gel electrophoresis was performed with replicate reactions to confirm that all reactions for all targets worked properly and produced significant amounts of amplification product. In reactions performed with the 4-base truncated target T3, the expected size shift was visible.

[0138] This example demonstrates how the first and second oligonucleotide probes, an essential feature of the present invention, can be used to provide not only rapid and sensitive detection of amplification products, but also to provide additional target sequence-based specificity checks of amplification products beyond those resulting from primer hybridization alone. This powerful technology overcomes known challenges of prior art methods resulting from non-target specific background amplification in certain assays due to synthesis from 1 or inter-primer binding. The method of the present invention demonstrates enhanced specificity compared to prior art methods, while maintaining sensitive detection and rapid, low-cost visualization of results.

[0139] Example 5 (Implementation of a method in which the moiety of the second oligonucleotide probe that allows binding to a solid material is an antigen and the corresponding antibody is bound to a solid surface, a nitrocellulose lateral flow strip) In the method of the invention, several different moieties can be utilized as moieties for binding the second oligonucleotide probe to a solid material. In this example, it is demonstrated that a method can be performed in which the moiety that allows binding of the second oligonucleotide probe to a solid material is an antigen, and the corresponding antibody is bound to a solid surface, a nitrocellulose lateral flow strip.

[0140] The second oligonucleotide probe was designed to contain a 32-base sequence that encompassed a region of homology to at least one species in the amplification product and a 3' digoxigenin NHS ester modification added during synthesis. Purified sheep Fab fragment anti-digoxigenin antibody (Sigma-Aldrich) was immobilized on the nucleic acid lateral flow strips by spotting and air drying.

[0141] The performance of the second oligonucleotide probe was evaluated by incubating various levels of target (+++=1 pmol; ++=0.1 pmol; +=10 fmol; NTC=no target control) at 0.016 mg ml -1 This was demonstrated in an experiment where carbon, containing carbon adsorbed to biotin-binding proteins, was added to 60 μl of a designed reaction buffer containing the necessary reagents for detection using a carbon nucleic acid lateral flow reaction. The strips were prepared by spotting 0.5 μg of anti-digoxigenin Fab fragments in 0.2 μl of a buffer containing 2.5 mM boric acid and 0.5% Tween 20 onto the strips. The solution was allowed to dry in the nitrocellulose membrane of the lateral flow strips for 2 hours. The reactions were incubated at 45° C. for 2 minutes to allow the formation of the designed detector species, after which the entire reaction mix of each reaction was applied to the lateral flow strips.

[0142] Figure 9 shows a photograph of the lateral flow strips produced in this experiment. Black spots corresponding to carbon deposits on the lateral flow strips are visible at each target level and not at the NTC, indicating that the detector species is specifically detected. The combination of a biotin-based affinity interaction for binding the detection moiety (carbon) and an antibody-based affinity interaction for the solid material binding moiety is shown. This example serves the purpose of demonstrating the versatility of this method in terms of the different approaches available for binding the second oligonucleotide probe to the solid material.

[0143] Example 6 (Implementation of the method, wherein the moiety enabling the second oligonucleotide probe to be bound to the solid material is a single stranded oligonucleotide comprising four repeated copies of a three base DNA sequence motif, and the reverse complement of said single stranded oligonucleotide sequence is bound to the solid material) This example shows the implementation of the method, in which the moiety that allows the second oligonucleotide probe to bind to the solid material is a single-stranded oligonucleotide that contains four repeated copies of a three-base DNA sequence motif. As mentioned above, the embodiment of the method that utilizes a single-stranded oligonucleotide as the detection moiety of the second oligonucleotide probe presents a simple and versatile aspect of the method that facilitates detection by nucleic acid lateral flow and easily realizes the detection of multiple different target nucleic acids in the same sample. Furthermore, the single-stranded oligonucleotide detection moiety can be predefined and optimized for efficient on-strip hybridization to enhance detection sensitivity and provide for efficient scale-up manufacturing of the nucleic acid lateral flow strip.

[0144] In one embodiment of the present invention, the inventors have observed an unexpected improvement in on-strip hybridization by the use of single-stranded oligonucleotide detection moieties composed of multiple repeated copies of a DNA sequence motif. In this example, the results of multiple side-by-side experiments are presented, in which the performance of an assay using a second oligonucleotide directly attached to a lateral flow strip is significantly enhanced by the use of a single-stranded detection moiety containing four repeated copies of a three-base DNA sequence motif, and the reverse complement of the single-stranded oligonucleotide sequence is attached to the lateral flow strip.

[0145] (Example 6.1) An assay was designed utilizing an embodiment of the method in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with a sample simultaneously with performing step a). A first oligonucleotide probe of 25 bases in length was designed that contains, in the 5' to 3' direction: a 5' biotin modification; a 7 base middle region; a 5 base restriction enzyme recognition site that is not a nicking enzyme; a 13 base region capable of hybridizing to a first hybridization region in a target that contains a phosphorothioate bond at the restriction enzyme cleavage site; and a 3' phosphate modification (the biotin modification allows the first oligonucleotide probe to be bound to a colorimetric dye, a carbon nanoparticle; the phosphate modification prevents its extension by a strand-displacing DNA polymerase).

[0146] Two alternative second oligonucleotide probes were designed to detect the same target species (I and II). The second oligonucleotide probe "I" was designed to contain in the 5' to 3' direction: 3x repeats of a 14 base region capable of hybridizing with the reverse complement of the second hybridization sequence in the target; and a 9x thymidine base spacer. Nucleic acid lateral flow strips were prepared with spots containing 30 pmol of probe.

[0147] An alternative second oligonucleotide probe "II" was designed, which includes in the 5' to 3' direction: a 14 base region capable of hybridizing with the reverse complement of the second hybridization region in the target; a 5x thymidine base intermediate spacer; and a 12 base single-stranded oligonucleotide portion including 4x repeats of a 3 base sequence motif that serves as a portion that allows the second oligonucleotide probe to bind to a solid material. An additional single-stranded oligonucleotide was designed to include in the 5' to 3' direction: an 11x thymidine base spacer; a 36 base region including 12x repeats of a reverse complement to a 3 base sequence motif that forms a portion that allows the second oligonucleotide II to bind to a solid material. For the second oligonucleotide probe II, a nucleic acid lateral flow strip was prepared, spotted with 30 pmol of the additional single-stranded oligonucleotide.

[0148] 0.016 mg / ml -1 Reactions to test the performance of oligonucleotide probes I and II were performed containing 0.5 pmol of the first oligonucleotide probe in 60 μl of the appropriate buffer containing carbon adsorbed to biotin-binding protein. Reactions II were similarly assembled but with the addition of 0.5 pmol of the second oligonucleotide probe II. Nucleic acid targets (single-stranded DNA targets representative of at least one species in the amplification products from the designed assay reagents) were added at various levels (+++=1 pmol, ++=0.1 pmol, NTC=no target control). The assembled reactions were incubated at 45°C for 2 minutes before the entire reaction mix was loaded onto the appropriate nucleic acid lateral flow strip.

[0149] Figure 10A shows a photograph of the lateral flow strips obtained in this experiment. The left panel displays the results with the second oligonucleotide probe I, and the right panel displays the results with the second oligonucleotide probe II. Black spots corresponding to the deposition of carbon-bound detector species were visualized in the presence of target. A stronger signal was observed at all target levels for the second oligonucleotide probe II, which contains a repeat sequence motif.

[0150] (Example 6.2) Next, to demonstrate the versatility of the above embodiment of the method and its wide applicability, separate assays were designed for completely different target nucleic acids. Oligonucleotide probes were designed for the related target nucleic acid, single-stranded DNA, in a similar manner as described in Example 6.1; again, the two formats of the second oligonucleotide probe were called "I" and "II" and various target levels (+++=1 pmol, ++=0.1 pmol, +=0.001 pmol) were used. Further striking effects were observed, as shown in the photograph of the produced lateral flow strips shown in Figure 10B. At the two lowest target levels tested, the second oligonucleotide probe I did not produce any signal, while the corresponding repeat sequence oligonucleotide probe II produced a clear positive signal indicated by a black spot of deposited carbon.

[0151] This example shows a significant improvement to lateral flow hybridization-based detection utilizing a second oligonucleotide detection moiety that contains repeated copies of a DNA sequence motif. It demonstrates that a 100-fold improvement in sensitivity of nucleic acid lateral flow-based detection of detector species can be obtained. Signal strength is enhanced and the signal develops more rapidly, demonstrating the potential of the above embodiment of the invention to facilitate application in applications involving rapid detection, for example, by nucleic acid lateral flow. It further illustrates the potential of using a single-stranded oligonucleotide as a detection moiety coupled to a second oligonucleotide probe.

[0152] Example 7 (Use of RNA virus detection methods in clinical specimens) In this example, we demonstrate the implementation of a method for detecting RNA viruses in clinical specimens using an embodiment of the method in which a first oligonucleotide probe is contacted with the sample simultaneously with the implementation of the amplification step a), and the portion that allows the second oligonucleotide probe to bind to the solid material is a single-stranded oligonucleotide that contains four repeated copies of a three-base DNA sequence motif, and the reverse complement of the single-stranded oligonucleotide sequence is bound to the solid material. In various studies, we routinely detect RNA targets with very low copy numbers, such as viral genome extracts. For example, using quantified viral genome extracts, we have utilized the method of the present invention to detect viruses with less than 100 genome equivalent copies in a total time to result of less than 10 minutes, with amplification step a) taking less than 5 minutes. This remarkable speed and sensitivity demonstrate the potential of the method for application in the field of diagnostics. Thus, in this example, we have developed an assay for detecting pathogenic single-stranded RNA viruses and demonstrated the performance of the assay using clinical specimens infected with the virus.

[0153] A first oligonucleotide primer was designed with a total length of 25 nucleotide bases, including in the 5' to 3' direction: an 8-base synthetic stabilization region with phosphorothioate bonds between each base; a 5-base non-nicking restriction enzyme recognition site; and a 12-base hybridization region with the reverse complement of the first hybridization sequence in the target nucleic acid, designed to target a region in the single-stranded RNA virus genome. A second oligonucleotide primer was designed with the same stabilization region, but without phosphorothioate bonds, and with the same restriction enzyme recognition sequence, but with a 12-base hybridization region that can hybridize with the reverse complement of the second hybridization sequence. In this example, the first restriction enzyme and the second restriction enzyme are the same restriction enzyme. The first and second hybridization sequences in the target nucleic acid are separated by 0 bases.

[0154] An oligonucleotide primer is designed using the target nucleic acid so that the nucleotide base downstream of the cleavage site of the reverse complement of the primer is adenosine, such that α-thiol dATP is utilized as the modified dNTP for use in the method. Phosphorothioate modifications are inserted by a strand-displacing DNA polymerase or reverse transcriptase to prevent cleavage of the reverse complement.

[0155] A first oligonucleotide probe of total length 24 bases was designed that contained in the 5' to 3' direction: a 5' biotin modification added during synthesis that allows binding of the first oligonucleotide probe to a colorimetric dye, a carbon nanoparticle; an 8-base stabilization region; a 5-base recognition sequence for a restriction enzyme that is not a nicking enzyme, where the cleavage site of said restriction enzyme in the first oligonucleotide probe is protected by a phosphorothioate internucleotide bond added during synthesis; an 11-base region that can hybridize to at least one species in the amplification product; and a 3' phosphate modification that prevents extension by a strand-displacing DNA polymerase.

[0156] A second oligonucleotide probe was designed with a total length of 31 bases, including: a 14-base region capable of hybridizing with a second single-stranded detection sequence downstream of the at least one species of first single-stranded detection sequence in the amplification product; a spacer including 5× thymidine bases; and 4× repeats of a three-base DNA sequence motif with its reverse complement immobilized on the lateral flow strip. An immobilized lateral flow printed oligonucleotide was designed with a total length of 47 bases, including: an intermediate spacer including 11× thymidine bases; and 12× repeats of a three-base sequence motif that is complementary to the three-base sequence motif of the second oligonucleotide probe. A lateral flow control oligonucleotide was designed with a length of 20 bases, including: a 5× triplet repeat sequence different from that on the second oligonucleotide probe; an intermediate spacer including 5× thymidine bases; and a 3′ biotin molecule added during synthesis. The control oligonucleotide binds to its reverse complement on the lateral flow strip and verifies whether the carbon lateral flow procedure is successful.

[0157] Reactions were prepared containing 1.8 pmol of first primer; 9.6 pmol of second primer; 3.6 pmol of first probe; 1 pmol of second probe; 300 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 28 U of restriction enzyme; 14 U of Bacillus strand-displacing DNA polymerase; 35 U of viral reverse transcriptase; 3.5 U of RNase H; and 3 μg of carbon adsorbed biotin-binding protein. 5 μl of nasopharyngeal swab samples collected from patients in a clinical setting (obtained from Discovery Life Sciences) included 7 virus-positive samples and 6 virus-negative clinical samples (verified by PCR assay). Reactions were performed in a volume of 70 μl in the appropriate reaction buffer. The reaction mixture was incubated at 45°C for 4 minutes and 30 seconds, after which the entire reaction mixture was loaded onto a nucleic acid lateral flow strip printed with approximately 50 pmol of the reverse complement of the 3-base triplet repeat portion of the second oligonucleotide probe (bottom line) and the reverse complement of a control oligonucleotide (top line).

[0158] Figure 11 shows a photograph of the lateral flow strip obtained in the implementation of this example. The arrows indicate the position (+) where the reverse complement of the triplet repeat sequence portion of the second oligonucleotide probe is printed, thus appearing as a positive signal, and the position of the reverse complement of the control oligonucleotide (CTL), verifying whether the lateral flow was successfully performed, thus appearing as both a positive and a negative assay. The top panel (+ve) shows the results obtained with a virus-positive clinical sample, and the bottom panel (-ve) with a virus-negative sample. A clear black line indicating the presence of the target nucleic acid was present in each of the positive samples, thereby demonstrating the rapid detection of clinical specimens by the method of the invention. No false positives were observed, demonstrating the complete absence of non-specific production of detector species, e.g. through synthesis from 1 or interprimer binding. No false negatives were observed, proving the robustness of the method and its sensitivity across different target nucleic acid copy number levels present in different clinical specimens.

[0159] Example 8 (Performing the method at different temperatures) The method of the present invention is efficiently carried out over a wide range of temperatures, and does not require temperature cycling, or any hot or warm start, preheating, or controlled temperature reduction. This example shows the performance of a typical assay over a range of different temperatures. By selecting an enzyme with a desired optimum temperature and using phosphorothioate bases that reduce the melting temperature of hybridization after incorporation, assays were easily developed in which amplification was carried out over an unexpected wide range of temperatures, usually covering a low temperature range. Furthermore, a separate experiment demonstrates that assays developed using the method of the present invention are developed without the need to preheat the sample before the start of step a), where no degradation in performance is observed when the temperature is increased during the performance of the amplification in step a).

[0160] (Example 8.1) An assay was designed utilizing an embodiment of the method in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with performing step a). A first primer was designed to include, in the 5' to 3' direction: a 7-base intermediate region; a recognition site for a restriction enzyme; and an 11-base region capable of hybridizing to a first hybridization sequence in a target nucleic acid, a DNA target. A second primer was designed to include, in the 5' to 3' direction: a 7-base intermediate region; a recognition site for the same restriction enzyme as the first primer; and a 12-base region capable of hybridizing to the reverse complement of a second hybridization sequence in a target nucleic acid.

[0161] A first oligonucleotide probe of 21 bases in length was designed to contain, in the 5' to 3' direction: a 5' biotin modification; a 6-base middle region; a recognition site base for a restriction enzyme containing a mismatch at position 2; a 10-base region capable of hybridizing to a first hybridization region in the target containing a G-clamp modification at position 6; and a 3' phosphate modification (the biotin modification allows the first oligonucleotide probe to be conjugated to a colorimetric dye, a carbon nanoparticle; the phosphate modification prevents its extension by strand-displacing DNA polymerase).

[0162] The second oligonucleotide probe was designed to include, in the 5' to 3' direction: an 11-base region capable of hybridizing with the reverse complement of the second hybridization sequence in the target; 12 bases including a 4x thymidine base spacer and a 4x repeat sequence of a 3-base sequence motif that serves as a portion that allows the second oligonucleotide probe to bind to a solid material. The additional single-stranded oligonucleotide was designed to include, in the 5' to 3' direction: an 11x thymidine base spacer; a 33-base region including an 11x repeat sequence of a reverse complement to a 3-base sequence motif that forms a portion that allows the second oligonucleotide to bind to a solid material. For the second oligonucleotide probe, a nucleic acid lateral flow strip was prepared on which 30 pmol of the additional single-stranded oligonucleotide was spotted.

[0163] Reactions were prepared in appropriate buffer containing 1.5 pmol of first primer; 1.0 pmol of second primer; 1 pmol of first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; and various levels of target DNA (++=1 amol, +=10 zmol, NTC=no target control). The assembled reactions were incubated at the target temperature (I=37° C.; II=45° C., III=50° C., and IV=55° C.) for 2 minutes before initiating the reaction by the addition of a final 5 U of restriction enzyme and 5 U of Bacillus strand-displacing DNA polymerase to a final reaction volume of 25 μl. The reactions were then incubated for 5 minutes (T1) or 8 minutes (T2) at the relevant target temperature. After incubation, each reaction was transferred to 75 μl of buffer containing 1.5 pmol of a second oligonucleotide probe and 8 μg of carbon adsorbed with biotin-binding protein, and then applied to the sample pad of a nucleic acid lateral flow strip.

[0164] Figure 12A shows photographs of lateral flow strips from experiments at each target level, temperature, and time point. The distinct black lines observed correspond to the deposition of carbon bound to the detector species produced in the presence of target. At all temperatures, very strong signals appeared in the presence of target at both target levels within 8 minutes, demonstrating the broad temperature range of efficient amplification by this method. No nonspecific amplification was observed in the NTC samples. Also, strong amplification was observed after only 5 minutes at 45°C and 50°C, indicating that the optimal temperature for this assay is likely to be between 40°C and 50°C.

[0165] (Example 8.2) A second assay was designed utilizing an embodiment of the method in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with a sample simultaneously with performing step a). Both a first and a second primer were designed that included, in the 5' to 3' direction: a 6 base intermediate region; a recognition site for a restriction enzyme; and a 12 base hybridization region for a target nucleic acid target. The primers were designed such that the first and second hybridization sequences in the target were separated by 10 bases.

[0166] A first oligonucleotide probe was designed with a total length of 23 bases, containing in the 5' to 3' direction: a 5' biotin modification; a 6-base middle region; a restriction enzyme base recognition site containing a mismatch at position 4; a 12-base region capable of hybridizing to a first hybridization region in the target; and a 3' phosphate modification (the biotin modification allows the first oligonucleotide probe to be conjugated to a colorimetric dye, a carbon nanoparticle; the phosphate modification prevents its extension by a strand-displacing DNA polymerase).

[0167] The second oligonucleotide probe was designed to include, in the 5' to 3' direction: a 13-base region capable of hybridizing with the 3-base reverse complement of the second hybridization sequence in the target and a 10-base gap between the first and second hybridization sequences; 12 bases including a 3x thymidine base spacer and a 4x repeat sequence of the 3-base sequence motif that serves as a portion that allows the second oligonucleotide probe to bind to a solid material. The additional single-stranded oligonucleotide was designed to include, in the 5' to 3' direction: an 11x thymidine base spacer; a 36-base region including a 12x repeat sequence of the reverse complement to the 3-base sequence motif that forms a portion that allows the second oligonucleotide probe to bind to a solid material. For the second oligonucleotide probe, a nucleic acid lateral flow strip was prepared on which 30 pmol of the additional single-stranded oligonucleotide was spotted.

[0168] Reactions were prepared in appropriate buffer containing 6 pmol of first oligonucleotide primer; 8 pmol of second oligonucleotide primer; 6 pmol of first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 60 μg of carbon adsorbed biotin-binding protein; and target, if applicable. The assembled reactions were incubated at the starting temperature (I=15° C.; II=45° C.) for 2 minutes before being initiated by the addition of a final 20 U of restriction enzyme, 20 U of Bacillus strand-displacing DNA polymerase, and 40 U of reverse transcriptase to a final reaction volume of 100 μl. After enzyme addition, the reaction with the starting temperature of 15° C. was immediately transferred to 45° C. along with the other reactions.

[0169] The reactions were then incubated at 45°C for 6 min. After incubation, each reaction was transferred to the sample pad of a nucleic acid lateral flow strip containing 3 pmol of the second oligonucleotide probe. Figure 12B shows a photograph of the lateral flow strip obtained in the experiment with each temperature incubation condition. The distinct black lines observed correspond to the deposition of carbon bound to the detector species produced in the presence of target. No differences were observed in the reactions in which the temperature was increased from 15°C to 45°C in the amplification step a). Significant amplification rates were obtained, comparable to the preheated reactions, and no nonspecific amplification was observed in the NTC samples.

[0170] This Example 8 demonstrates that the method of the present invention can be used to easily develop assays that have a lower optimal temperature profile compared to known methods and can utilize highly sensitive detection over an exceptionally wide temperature range. Also, the method of the present invention can be performed without preheating to increase the temperature in the performance of step a). Such features make the method very attractive for use in low-cost diagnostic devices, where high temperatures and strict heating control impose complex physical constraints that increase the cost of goods for such devices to the point where disposable or instrument-free devices are commercially impractical. Furthermore, by avoiding the requirement of known methods to preheat the sample before amplification begins, the method of the present invention can be performed in a more simplified sequence of operations with fewer user steps, thus improving the ease of use of such diagnostic devices and reducing the overall time to result.

[0171] Example 9 (Implementation of a method in which a target nucleic acid is obtained from double-stranded DNA by strand invasion) This example demonstrates the use of this method where the single-stranded target nucleic acid is a single-stranded site in double-stranded DNA that is detected without any requirement for any specific actions to separate the DNA strands, such as temperature denaturation, bump primers, or the use of additional enzymes (e.g., helicases or recombinases). The ability to readily detect both single-stranded RNA and double-stranded DNA targets using the method of the invention without additional user steps, components, or physical requirements imposed on the equipment used to perform the method, confers this method a high degree of versatility for use in diagnostic applications.

[0172] Example 9.1: An assay was developed for protein coding regions in double-stranded DNA genomes of viral targets. Double-stranded genomes or mRNA transcripts can be used as biomarkers for the presence of viruses in clinical diagnostics. The assay was designed using an embodiment of the method in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with the performance of step a). The design of the oligonucleotide primers and oligonucleotide probes was performed following an approach similar to that described in the other examples, without a gap between the first and second hybridization sequences in the target nucleic acid.

[0173] Reactions were prepared in appropriate buffer containing 4 pmol of first oligonucleotide primer; 2 pmol of second oligonucleotide primer; 2 pmol of first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 60 μg of carbon adsorbed to biotin-binding protein; and double-stranded DNA target (I) or single-stranded RNA target (II) or no target. The assembled reactions were incubated at 45° C. for 2 minutes before being initiated by the addition of a final 20 U of restriction enzyme, 20 U of Bacillus strand-displacing DNA polymerase, and 25 U of reverse transcriptase to a final reaction volume of 100 μl. After addition of the enzymes, the reactions were incubated at 45° C. for 7 minutes.

[0174] After incubation, 1.5 pmol of the second oligonucleotide probe was added to each reaction and the entire reaction volume was transferred to the sample pad of a nucleic acid lateral flow strip. A nucleic acid lateral flow control target was also added to all samples. Figure 13A shows a photograph of the lateral flow strip obtained in this experiment for each target. The distinct black lines observed correspond to the deposition of carbon bound to the detector species produced in the presence of target. The fainter signals correspond to lower target levels (+) than higher target levels (++). No difference in amplification rates was observed between single-stranded RNA and double-stranded DNA targets.

[0175] (Example 9.2) An assay was designed for a single-stranded target nucleic acid in the c.2.5 megabase double-stranded DNA genome of a bacterial pathogen. The assay was designed utilizing an embodiment of the method in which the first oligonucleotide probe is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with performing step a). The oligonucleotide primers and oligonucleotide probes were designed following an approach similar to that described in the other examples, with a four base gap between the first and second hybridization sequences in the target nucleic acid.

[0176] Reactions were prepared in a suitable buffer containing 2 pmol of the first oligonucleotide primer and 0.5 pmol of the second oligonucleotide primer. Because a double-stranded DNA target is used, it is assumed that in fact two single-stranded target nucleic acids are added simultaneously and a second reciprocal process occurs at the same time. Here, the second oligonucleotide primer for detection of the target nucleic acid is the first oligonucleotide primer for detection of the second target nucleic acid, which is the reverse complement of the target nucleic acid. This fact has little effect on the performance of the method. 2 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 15 μg carbon adsorbed to biotin-binding protein; and bacterial genomic extracts containing the target at various levels (++=1 amol; +=10 zmol; NTC=no target control). In addition, a specificity control reaction was also performed containing 1 amol of E. coli genomic extract. The assembled reactions were incubated at 45°C for 3 minutes and then initiated by the addition of a final 4 U of restriction enzyme and 2 U of Bacillus strand-displacing DNA polymerase to a final reaction volume of 25 μl. After addition of the enzymes, the reactions were incubated at 45°C for 6 minutes.

[0177] After incubation, 75 μl of buffer containing 3 pmol of the second oligonucleotide probe was added to each reaction, and the entire volume was then transferred to the sample pad of the nucleic acid lateral flow strip. Figure 13B shows a photograph of the lateral flow strip obtained in this experiment for each target. A clear black line corresponding to the deposition of carbon bound to the detector species produced in the presence of target was observed at both target levels tested. No nonspecific signal was observed in the no target control or in the presence of E. coli genomic DNA, demonstrating that this method can be used for the specific detection of complex double-stranded DNA genomes at clinically relevant copy numbers within just 6 minutes.

[0178] This example demonstrates that the method of the present invention can be easily used to detect single-stranded nucleic acid targets in double-stranded DNA. Notably, comparable amplification rates were observed for detection of single-stranded RNA targets and the same target sequence in double-stranded DNA without the requirement of any specific action to separate the DNA duplex, such as temperature denaturation. Instead, the single-stranded site is sufficiently exposed for hybridization and extension of the first oligonucleotide primer, initiating the method by "strand invasion." Here, a transient opening of one or more DNA base pairs in the double-stranded DNA occurs sufficient to allow hybridization and extension of the 3' hydroxyl of the first oligonucleotide primer. This is in contrast to known methods such as SDA that utilize thermal denaturation and bump primers in assays of double-stranded nucleic acids. The ability to use the method of the present invention to easily detect targets in double-stranded DNA as well as in single-stranded DNA and single-stranded RNA confers a high degree of versatility for its diagnostic applications, such as use in detecting bacterial, fungal, and viral pathogens that have double-stranded DNA genomes. The fact that this method does not require complex additional user steps, enzymes, components, or physical constraints to detect organisms with double-stranded DNA genomes means that this method is particularly well suited for testing with simplified, low-cost diagnostic devices. For example, the requirement of heat denaturation before performing amplification reported for known methods requires expensive additional components, increasing the cost of goods and total time to result for such devices. This means that disposable or self-contained, instrument-free devices are not practical.

[0179] Example 10 (Comparison of the method of the present invention with known methods) This example presents a comparative evaluation of the method of the invention against the known method disclosed in WO2014 / 164479 for detecting viral targets. The known method is fundamentally different from the method of the invention in that it requires a nicking enzyme and does not require the use of one or more modified dNTPs. The method of the invention demonstrates exceptional sensitivity and specificity.

[0180] For this comparative evaluation, an assay for a viral target having a single-stranded RNA genome was first developed using the method of the present invention. The assay was designed using an embodiment of the method in which the first oligonucleotide probe is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and the probe is contacted with the sample simultaneously with the performance of step a). The oligonucleotide primers and oligonucleotide probes were designed following an approach similar to that described in the other examples, with a 6-base gap between the first and second hybridization sequences in the target nucleic acid.

[0181] For the assay using known methods, similar primers were designed, containing the same 6-base intermediate region at the 5' end and the same hybridization region at the 3' end as the equivalent primers used in the method of the present invention.In this way, the consistency between the two assays was ensured as much as possible in order to perform a strict comparison of the methods.However, the bases of the restriction enzyme recognition site were replaced with the bases of the recognition site of an exemplary nicking enzyme reported in WO2014 / 164479, Nt.BbvCI (see Example 5 on p.20-21).

[0182] (Example 10.1) In the first example, reactions for each method were performed using equal primer ratios. For the method of the present invention, reactions were prepared in appropriate buffers containing: 2 pmol of first oligonucleotide primer; 2 pmol of second oligonucleotide primer; 1.6 pmol of first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; and various levels of viral genomic RNA extract as target (+++=10 zmol; ++=100 copies; +=10 copies; NTC=no target control). The assembled reactions were pre-incubated at ambient temperature (c. 20° C.) for 5 minutes, and then initiated by the addition of 5 U of restriction enzyme, 5 U of Bacillus strand-displacing DNA polymerase, and 10 U of reverse transcriptase to a final reaction volume of 25 μl. After addition of the enzymes, the reactions were incubated at 45° C. for 8 minutes (T1) or 15 minutes (T2). After incubation, each reaction received additional 60 μg of carbon adsorbed with biotin-binding protein in 75 μl buffer, and the total 100 μl volume was transferred to a nucleic acid lateral flow strip containing 1.5 pmol of a second oligonucleotide probe on the sample pad.

[0183] For known methods, reactions were prepared in appropriate buffers containing: 6.25 pmol of the first oligonucleotide primer; 6.25 pmol of the second oligonucleotide primer; 200 μM each of dATP, dTTP, dCTP, and dGTP; and various levels of viral genomic RNA extract as target (+++=10 zmol; ++=100 copies; +=10 copies; NTC=no target control). The assembled reactions were preincubated at ambient temperature (c. 20° C.) for 5 minutes before being initiated by the addition of 4 U of Nt.BbvCI, 20 U of Bst large fragment DNA polymerase, and 10 U of M-MuLV reverse transcriptase to a final reaction volume of 25 μl. After addition of the enzymes, the reactions were incubated at 45° C. for 8 minutes (T1) and 15 minutes (T2). After incubation, 75 μl buffer containing 60 μg of biotin-binding protein-adsorbed carbon and 5 pmol of the first oligonucleotide probe was added to each reaction, and the total 100 μl volume was transferred to a nucleic acid lateral flow strip containing 5 pmol of the second oligonucleotide probe on the sample pad.

[0184] In FIG. 14A, photographs of lateral flow strips obtained in experiments using the method of the present invention (I) and known method (II) at various target levels and indicated time points are shown. The observed black lines correspond to carbon deposits bound to detector species produced in the presence of target. Several trials were required and specific combinations of enzymes and buffers, as well as significantly higher levels of primers, dNTPs, and enzymes, were required before any signal could be observed using known methods. Using the method of the present invention (I), the detector species produced could be clearly identified even at the lowest target levels, with only 10 copies of target at the shortest time point after only 8 minutes without preheating. Even after efforts to optimize known methods that were not obvious to those skilled in the art, only faint signals were observed at the highest target levels (+++=10 zmol) and the longest time point (15 minutes).

[0185] (Example 10.2) Furthermore, after extensive and non-obvious attempts, it was possible to improve the performance of the known method, but only by using a 2:1 ratio of first and second oligonucleotide primers, with a very high concentration of the first primer, as described in this Example 10.2. The method of the invention was again carried out as described in Example 10.1. For the known method, the reaction was carried out as described in Example 10.1, except that the level of the first oligonucleotide primer was increased to 12.5 pmol. In each case, the following target levels were used: +++=1 zmol; ++=100 copies; +=10 copies; NTC=no target control.

[0186] In FIG. 14B, photographs of lateral flow strips obtained in experiments using the method of the present invention (I) and known method (II) at various target levels and indicated time points are shown. The observed black lines correspond to the deposition of carbon bound to detector species produced in the presence of target. Here again, the method of the present invention (I) was demonstrated to obtain remarkable speed with visible signals even at very low target levels of only 10 copies of target at the shortest time point. With known methods, only faint signals were observed at the highest target levels (+++=1 zmol), and very faint signals were visible even at the longest time point (15 min) in the 100 copy sample. However, faint signals were also observed in NTC strips that could correspond to non-specific products as a result of the very high oligonucleotide primer and enzyme levels required for the method to function. These data are consistent with those in WO2014 / 164479, which reported an incubation time of 30 min. The requirement for the addition of exceptionally high primer levels to speed up amplification performed using this known method severely limits its potential applicability for the detection of two or more different targets in the same sample, since there is very limited scope for further increasing the total primer levels without exacerbating the problems with non-specific products.

[0187] This Example 14 demonstrates the remarkable superiority of the method of the present invention over the known method disclosed in WO2014 / 164479, in that amplification is performed much faster, more sensitive, and produces a clearer result signal.Without pre-incubation, in only 8 minutes, the method of the present invention produces a stronger signal than can be produced in the known method, which can be performed in 15 minutes at the highest target level using only 100 copies of target and 60 times the target level.The advantage of the method of the present invention over this known method comes from the fact that it requires a different class of enzyme, which is a restriction enzyme that is not a nicking enzyme, and that it requires the use of one or more modified dNTPs, such as phosphorothioate bases, which enhance the sensitivity and specificity of amplification. Further, an embodiment of the method, wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and is contacted with the sample simultaneously with the performance of step a), allowing efficient coupling of amplification with signal detection and promoting enhanced specificity resulting from efficient sequence-based hybridization during the formation of detector species. These advantages make the method of the present invention ideally suited for use in the field of diagnostics and for the development of simplified, ultrafast, user-focused, low-cost diagnostic devices, such as disposable or instrument-free molecular diagnostic test devices. Throughout the following specification and claims, unless the context otherwise requires, it will be understood that the terms "comprise", and variations such as "comprises", "comprising" and the like imply the inclusion of the stated integer, step, group of integers, or group of steps, but do not exclude any other integer, step, group of integers, or group of steps.

[0188] Further aspects of the present invention include the following: Embodiment 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by a DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by a DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) subjecting the amplification product of step a) to: i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. contacting with a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product and attached to a solid material or a moiety that allows for binding to the solid material, wherein hybridization of the first and second probes to the at least one species in the amplification product produces a detector species; and c) detecting the presence of the detector species produced in step b), the presence of the detector species indicating the presence of the target nucleic acid in the sample. Aspect 2. The method of aspect 1, wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme. Aspect 3. The method of aspect 2, wherein said one oligonucleotide probe cannot be cleaved by said first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate linkages. Aspect 4. The method according to aspect 2 or 3, wherein said one oligonucleotide probe is contacted with said sample simultaneously with carrying out step a). Aspect 5. The method of any one of Aspects 1 to 4, wherein in step a) the sample is further contacted with: (A) a third oligonucleotide primer which comprises, in a 5' to 3' direction, a recognition sequence and cleavage site for the first restriction enzyme and one strand of a region capable of hybridizing to the first hybridization sequence in the target nucleic acid, and wherein extension by a DNA polymerase is blocked at its 3' end; and / or (B) a fourth oligonucleotide primer which comprises, in a 5' to 3' direction, a recognition sequence and cleavage site for the second restriction enzyme and one strand of a region capable of hybridizing to the reverse complement of the second hybridization sequence in the target sequence, and wherein extension by a DNA polymerase is blocked at its 3' end. Embodiment 6. The method of embodiment 5, wherein said third oligonucleotide primer, if present, is provided in excess of said first oligonucleotide primer, and said fourth oligonucleotide primer, if present, is provided in excess of said second oligonucleotide primer. Embodiment 7. The method according to any one of embodiments 1 to 6, wherein the one or more modified dNTPs are α-thiol modified dNTPs. Aspect 8. The method according to any one of aspects 1 to 7, wherein the first and second restriction enzymes are the same restriction enzyme. Embodiment 9. The method according to any one of embodiments 1 to 8, wherein two or more of steps a), b), and c) are carried out simultaneously. Embodiment 10. The method of any one of embodiments 1 to 9, wherein step (a) is carried out at a temperature of 50° C. or less. Aspect 11. The method of any one of aspects 1 to 10, wherein the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorometric dye, or a moiety capable of binding to a colorimetric or fluorometric dye, such as biotin. Embodiment 12. The method of any one of embodiments 1 to 11, wherein said detector species is detected by a change in an electrical signal. Embodiment 13. The method of any one of embodiments 1 to 12, wherein the moiety enabling detection of the first oligonucleotide probe is an enzyme that generates a detectable signal, such as a colorimetric or fluorometric signal, after contact with a substrate. Embodiment 14. The method according to any one of embodiments 1 to 13, wherein the moiety of the second oligonucleotide probe that enables binding to a solid material is a single-stranded oligonucleotide. The method of embodiment 14, wherein the sequence of the single-stranded oligonucleotide portion comprises three or more repeated copies of a two- to four-base DNA sequence motif. Embodiment 16. The method according to any one of embodiments 1 to 15, wherein in step c) the presence of said detector species is detected by nucleic acid lateral flow. Embodiment 17. The method of embodiment 16, wherein said nucleic acid lateral flow utilizes one or more nucleic acids capable of sequence-specific hybridization with a moiety that enables binding of said second oligonucleotide probe to a solid material. Embodiment 18. The method according to any one of embodiments 1 to 17, wherein step c) generates a colorimetric or electrochemical signal using carbon or gold, preferably carbon. Embodiment 19. The method according to any one of embodiments 1 to 18, wherein the first and / or second oligonucleotide primer comprises a stabilizing sequence, e.g., 5 bases long, at the 5' end upstream of the restriction enzyme recognition sequence and cleavage site. Aspect 20. The method according to any one of aspects 1 to 19, wherein the hybridizing region of the first and / or second oligonucleotide primer is 9 to 16 bases in length. Embodiment 21. The method of any one of embodiments 1 to 20, wherein one of the first and second oligonucleotide primers is provided in excess of the other primer. Aspect 22. The method of any one of aspects 1 to 21, wherein the first and second hybridization sequences in the target nucleic acid are separated by 0 to 6 bases. Aspect 23. The method of any one of aspects 1 to 21, wherein the first and second hybridization sequences in the target nucleic acid are separated by 3 to 6 bases. Aspect 24. The method according to any one of aspects 1 to 23, wherein in step b), the first or second single-stranded detection sequence of the at least one species in the amplification product comprises a sequence corresponding to 3 to 6 bases as defined in aspect 23. Embodiment 25. The method according to any one of embodiments 1 to 24, wherein the level of the target nucleic acid in the sample is quantified in step c). Aspect 26. The method according to any one of Aspects 1 to 25, wherein the target nucleic acid is single-stranded RNA, including single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA, or single-stranded DNA, including single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA. Embodiment 27. The method according to embodiment 26, wherein said single-stranded DNA is obtained from double-stranded DNA by use of a nuclease, such as a restriction endonuclease or exonuclease III, or from single-stranded RNA by use of a reverse transcriptase. Embodiment 28. The method according to any one of embodiments 1 to 27, wherein the presence of two or more different target nucleic acids of defined sequence is detected in the same sample. Embodiment 29. The method of any one of embodiments 1 to 28, wherein said sample is a biological sample, such as a nasal or nasopharyngeal swab or aspirate, blood or a blood-derived sample, or urine. Aspect 30. The method of any one of aspects 1 to 29, wherein the target nucleic acid is viral or derived from viral nucleic acid material, bacterial or derived from bacterial nucleic acid material, circulating cell-free DNA released from cancer cells or fetal cells, or is a microRNA or derived from a microRNA. Embodiment 31. The method of any one of embodiments 1 to 30, wherein the target nucleic acid comprises a site of epigenetic modification, such as methylation. Embodiment 32. The method according to any one of embodiments 1 to 31, wherein detection of said target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or disease state. Aspect 33. The method of aspect 32, wherein said disease is an infectious disease, including, but not limited to, HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox. Aspect 34. The method of aspect 32, wherein said disease is cancer, including but not limited to colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma. Aspect 35. The method according to any one of aspects 1 to 34, wherein detection of the target nucleic acid is used for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics. Embodiment 36 The method according to any one of embodiments 1 to 35, wherein the sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample. Embodiment 37. A kit comprising: a) a first oligonucleotide primer and a second oligonucleotide primer, the first primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to a first hybridization sequence in a single-stranded target nucleic acid of defined sequence, and the second primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; b) a first restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the first primer and cleaving the cleavage site, and a second restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the second primer and cleaving the cleavage site; c) strand-displacing DNA polymerase; d) dNTPs, e) one or more modified dNTPs; f) a first oligonucleotide probe having a degree of complementarity with the hybridizing region of one of the first and second oligonucleotide primers and attached to a moiety that allows for its detection; and g) a second oligonucleotide probe having a degree of complementarity with the reverse complement of the hybridizing region of the other of the first and second oligonucleotide primers and attached to a solid material or a moiety that enables binding to the solid material. Embodiment 38. The kit of embodiment 37, further comprising a means for detecting the presence of the detector species. Aspect 39. The kit according to aspect 37 or 38, wherein the first oligonucleotide primer, and / or the second oligonucleotide primer, and / or the first restriction enzyme, and / or the second restriction enzyme, and / or the DNA polymerase, and / or the dNTPs, and / or the one or more modified dNTPs, and / or the first oligonucleotide probe, and / or the second oligonucleotide probe are as defined in any one of aspects 2, 3, 7, 8, 11, 13 to 17, 19, 20, or 22 to 24. Embodiment 40. The kit according to any of embodiments 37 to 39, further comprising a third and / or a fourth oligonucleotide primer as defined in embodiment 5 or 6. Embodiment 41. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) Purifying the amplification product of step a): i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. contacting with a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product and attached to a solid material or a moiety that allows for binding to the solid material, wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and hybridization of the first and second probes with the at least one species in the amplification product produces a detector species; and c) detecting the presence of the detector species produced in step b), the presence of the detector species indicating the presence of the target nucleic acid in the sample; wherein said first or second oligonucleotide probe as defined in step b) is contacted with said sample simultaneously with carrying out step a).

[0189] All patents and patent applications mentioned herein are incorporated by reference in their entirety. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and the second primer comprises, in a 5' to 3' direction, a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the first primer and is capable of cleaving only the first primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; and vi. a second restriction enzyme that is not a nicking enzyme but that recognizes the recognition sequence of the second primer and is capable of cleaving only the second primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complement is prevented by the presence of one or more modifications incorporated into the reverse complement by the DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the target nucleic acid without the use of temperature cycling; b) Purifying the amplification product of step a): i. a first oligonucleotide probe capable of hybridizing to and allowing detection of at least one species of a first single-stranded detection sequence in the amplification product, the first oligonucleotide probe being linked to a moiety that allows detection thereof; and ii. a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; wherein hybridization of the first and second probes with the at least one species in the amplification product produces a detector species; and c) detecting the presence of the detector species produced in step b), the presence of the detector species being indicative of the presence of the target nucleic acid in the sample; The method comprising: (Aspect 2) 2. The method of embodiment 1, wherein one of the first and second oligonucleotide probes is blocked at its 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme. (Aspect 3) 3. The method of embodiment 2, wherein the blocked oligonucleotide probe cannot be cleaved by the first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate linkages. (Aspect 4) The method of embodiment 2 or 3, wherein said blocked oligonucleotide probe is contacted with said sample simultaneously with performing step a). (Aspect 5) The method according to any one of aspects 2 to 4, wherein the blocked oligonucleotide probe comprises an additional region, such that the 3' end of the species in the amplification product to which the blocked oligonucleotide probe hybridizes can be extended by the strand-displacing DNA polymerase. (Aspect 6) 6. The method according to any one of aspects 1 to 5, wherein in step a) the sample is further contacted with: (A) a third oligonucleotide primer which comprises, in a 5' to 3' direction, a recognition sequence and cleavage site for the first restriction enzyme and one strand of a region capable of hybridizing to the first hybridization sequence in the target nucleic acid, and wherein extension by a DNA polymerase is blocked at its 3' end; and / or (B) a fourth oligonucleotide primer which comprises, in a 5' to 3' direction, a recognition sequence and cleavage site for the second restriction enzyme and one strand of a region capable of hybridizing to the reverse complement of the second hybridization sequence in the target nucleic acid, and wherein extension by a DNA polymerase is blocked at its 3' end. (Aspect 7) 6. The method of embodiment 5, wherein said third oligonucleotide primer, if present, is provided in excess of said first oligonucleotide primer, and said fourth oligonucleotide primer, if present, is provided in excess of said second oligonucleotide primer. (Aspect 8) The method according to any one of aspects 1 to 7, wherein the one or more modified dNTPs are α-thiol modified dNTPs. (Aspect 9) The method according to any one of aspects 1 to 8, wherein the first and second restriction enzymes are the same restriction enzyme. (Aspect 10) The method according to any one of embodiments 1 to 9, wherein two or more of steps a), b), and c) are carried out simultaneously. (Aspect 11) The method according to any one of embodiments 1 to 10, wherein step a) is carried out at a temperature of 50° C. or lower. (Aspect 12) The method according to any one of the preceding aspects, wherein the temperature is increased during the performance of step a), such as increasing from an initial ambient temperature, for example in the range of 15-30°C, to a temperature in the range of 40-50°C. (Aspect 13) 13. The method of any one of aspects 1 to 12, wherein the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorometric dye, or a moiety capable of binding to a colorimetric or fluorometric dye, such as biotin. (Aspect 14) The method according to any one of embodiments 1 to 13, wherein the detector species is detected by a change in an electrical signal. (Aspect 15) The method according to any one of embodiments 1 to 14, wherein the detectable moiety of the first oligonucleotide probe is an enzyme that generates a detectable signal, such as a colorimetric or fluorescent signal, after contact with a substrate. (Aspect 16) The method according to any one of aspects 1 to 15, wherein the moiety of the second oligonucleotide probe that enables binding to a solid material is a single-stranded oligonucleotide. (Aspect 17) The method of embodiment 16, wherein the sequence of the single-stranded oligonucleotide portion comprises three or more repeated copies of a two- to four-base DNA sequence motif. (Aspect 18) The method according to any one of embodiments 1 to 17, wherein in step c) the presence of said detector species is detected by nucleic acid lateral flow. (Aspect 19) 20. The method of embodiment 18, wherein said nucleic acid lateral flow utilizes one or more nucleic acids capable of sequence-specific hybridization with a moiety that enables binding of said second oligonucleotide probe to a solid material. (Aspect 20) The method according to any one of embodiments 1 to 19, wherein step c) generates a colorimetric or electrochemical signal using carbon or gold, preferably carbon. (Aspect 21) The method according to any one of aspects 1 to 20, wherein the first and / or second oligonucleotide primer comprises a stabilizing sequence, for example, 5 or 6 bases long, upstream of the restriction enzyme recognition sequence and the cleavage site. (Aspect 22) 22. The method according to any one of aspects 1 to 21, wherein the hybridizing region of the first and / or second oligonucleotide primer is 9 to 16 bases in length. (Aspect 23) 23. The method of any one of aspects 1 to 22, wherein one of the first and second oligonucleotide primers is provided in excess of the other primer. (Aspect 24) 24. The method according to any one of aspects 1 to 23, wherein the first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 bases. (Aspect 25) 25. The method according to any one of aspects 1 to 24, wherein the first and second hybridization sequences in the target nucleic acid are separated by 3 to 15 bases. (Aspect 26) 26. The method according to any one of aspects 1 to 25, wherein in step b), the at least one species of first or second single-stranded detection sequence in the amplification product comprises a sequence of at least 3 bases corresponding to the 3 to 15 bases defined in aspect 24. (Aspect 27) The method according to any one of embodiments 1 to 26, wherein the level of said target nucleic acid in said sample is quantified in step c). (Aspect 28) The method according to any one of aspects 1 to 27, wherein the target nucleic acid is single-stranded RNA, including single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA, or single-stranded DNA, including single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA. (Aspect 29) 30. The method of embodiment 28, wherein the single-stranded target nucleic acid is single-stranded DNA obtained from double-stranded DNA by strand invasion. (Aspect 30) 30. The method of embodiment 28, wherein said single-stranded DNA is obtained from double-stranded DNA by use of a nuclease, such as a restriction endonuclease or exonuclease III, or from single-stranded RNA by use of a reverse transcriptase. (Aspect 31) The method according to any one of embodiments 1 to 30, wherein the presence of two or more different target nucleic acids of defined sequence is detected in the same sample. (Aspect 32) 32. The method according to any one of aspects 1 to 31, wherein the sample is a biological sample, such as a nasal or nasopharyngeal swab or aspirate, blood or a blood-derived sample, or urine. (Aspect 33) 33. The method of any one of aspects 1 to 32, wherein the target nucleic acid is viral or derived from viral nucleic acid material, bacterial or derived from bacterial nucleic acid material, circulating cell-free DNA released from cancer cells or fetal cells, or is a microRNA or derived from a microRNA. (Aspect 34) The method according to any one of aspects 1 to 33, wherein detection of the target nucleic acid is used for diagnosing, prognosing or monitoring a disease or disease state. (Aspect 35) 35. The method of embodiment 34, wherein said disease is an infectious disease, including, but not limited to, HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox. (Aspect 36) The method of embodiment 34, wherein said disease is cancer, including but not limited to colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma. (Aspect 37) The method according to any one of the preceding aspects, wherein detection of the target nucleic acid is used for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics. (Aspect 38) 38. The method according to any one of aspects 1 to 37, wherein the sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample. (Aspect 39) A kit comprising: a) a first oligonucleotide primer and a second oligonucleotide primer, the first primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to a first hybridization sequence in a single-stranded target nucleic acid of defined sequence, and the second primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; b) a first restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the first primer and cleaving the cleavage site, and a second restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the second primer and cleaving the cleavage site; c) strand-displacing DNA polymerase; d) dNTPs, e) one or more modified dNTPs; f) a first oligonucleotide probe that is capable of hybridizing to and permitting detection of at least one species of a first single-stranded detection sequence in an amplification product produced in the presence of the target nucleic acid and that is linked to a moiety that permits its detection; and g) a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product and attached to a solid material or a moiety that enables binding to the solid material. (Aspect 40) 40. The kit of embodiment 39, wherein one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the DNA polymerase and cannot be cleaved by the first or second restriction enzyme, e.g. due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate bonds. (Aspect 41) 41. The kit of aspect 39 or 40, wherein one of the first and second oligonucleotide probes has 5 or more bases of complementarity with the hybridizing region of the first or second primer, or the reverse complement of the hybridizing region of the first or second primer. (Aspect 42) 42. The kit of embodiment 41, wherein the first oligonucleotide probe has 5 or more bases of complementarity with a hybridizing region of one of the first and second oligonucleotide primers, and the second oligonucleotide probe has 5 or more bases of complementarity with a reverse complement of a hybridizing region of the other of the first and second oligonucleotide primers. (Aspect 43) 43. The kit of any one of embodiments 39 to 42, further comprising a means for detecting the presence of a detector species produced in the presence of said target nucleic acid. (Aspect 44) 44. The kit according to any one of aspects 39 to 43, wherein the target nucleic acid, the first oligonucleotide primer, and / or the second oligonucleotide primer, and / or the first restriction enzyme, and / or the second restriction enzyme, and / or the DNA polymerase, and / or the dNTPs, and / or the one or more modified dNTPs, and / or the first oligonucleotide probe, and / or the second oligonucleotide probe, and / or the first or second single-stranded detection sequence in at least one species in the amplification product are as defined in any one of aspects 5, 8, 9, 13, 15 to 17, 21 to 26, or 28 to 33. (Aspect 45) 45. The kit according to any of embodiments 39 to 44, further comprising a third and / or a fourth oligonucleotide primer as defined in embodiment 5 or 6. (Aspect 46) A device comprising the kit according to any one of embodiments 39 to 45. (Aspect 47) 47. The device of embodiment 46, which is a powered device. (Aspect 48) 48. The apparatus of embodiment 46 or 47, comprising a heating means. (Aspect 49) The device of any one of embodiments 46 to 48, which is a disposable diagnostic device.

Claims

1. 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i) a first oligonucleotide primer and a second oligonucleotide primer, the first oligonucleotide primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to a first hybridization sequence in the single-stranded target nucleic acid, and the second oligonucleotide primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and one strand of a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the single-stranded target nucleic acid; ii) strand-displacing DNA polymerase; iii) dNTPs; iv) one or more modified dNTPs; v) a first restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the first oligonucleotide primer and cleaving only the first oligonucleotide primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complementary strand is prevented by the presence of one or more modifications incorporated into the reverse complementary strand by the strand displacing DNA polymerase using the one or more modified dNTPs; and vi) a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second oligonucleotide primer and cleaving only the second oligonucleotide primer strand at the cleavage site when the recognition sequence and the cleavage site are double stranded, wherein cleavage of the reverse complementary strand is prevented by the presence of one or more modifications incorporated into the reverse complementary strand by the strand displacing DNA polymerase using the one or more modified dNTPs; to produce an amplification product in the presence of the single-stranded target nucleic acid without the use of temperature cycling; b) PCR using the amplification product of step a): i) a first oligonucleotide probe capable of hybridizing to and linking to a moiety enabling detection of at least one species of a first single-stranded detection sequence in the amplification product; and ii) a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in the same strand of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; contacting the At least one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the strand-displacing DNA polymerase and cannot be cleaved by the first or second restriction enzyme, and is contacted with the sample simultaneously with carrying out step a); said contacting producing a detector species upon hybridization of said first and second probes with said at least one species in said amplification product; and c) detecting the presence of the detector species produced in step b), the presence of the detector species being indicative of the presence of the single stranded target nucleic acid in the sample; The method comprising:

2. 2. The method of claim 1, wherein at least one of the first and second oligonucleotide probes is blocked at its 3' end from extension by the strand-displacing DNA polymerase and cannot be cleaved by the first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications to the restriction enzyme recognition sequence.

3. 3. The method of claim 2, wherein said one or more modifications are phosphorothioate linkages.

4. The method according to any one of claims 1 to 3, wherein at least one of the first and second oligonucleotide probes blocked at their 3' ends from extension by the strand-displacing DNA polymerase comprises an additional region, such that the 3' end of the at least one species in the amplification product to which the blocked oligonucleotide probe hybridizes can be extended by the strand-displacing DNA polymerase.

5. The method according to any one of claims 1 to 4, wherein in step a) the sample is further contacted with: (A) a third oligonucleotide primer, the third oligonucleotide primer comprising, in a 5' to 3' direction, a recognition sequence and a cleavage site for the first restriction enzyme and one strand of a region capable of hybridizing to the first hybridization sequence in the single-stranded target nucleic acid, and the extension by the strand-displacing DNA polymerase is blocked at the 3' end; and / or (B) a fourth oligonucleotide primer, the fourth oligonucleotide primer comprising, in a 5' to 3' direction, a recognition sequence and a cleavage site for the second restriction enzyme and one strand of a region capable of hybridizing to the reverse complement of the second hybridization sequence in the single-stranded target nucleic acid, and the extension by the strand-displacing DNA polymerase is blocked at the 3' end.

6. 6. The method of claim 5, wherein said third oligonucleotide primer, if present, is provided in excess of said first oligonucleotide primer.

7. 6. The method of claim 5, wherein the fourth oligonucleotide primer, if present, is provided in excess of the second oligonucleotide primer.

8. The method of any one of claims 1 to 7, wherein the one or more modified dNTPs are alpha thiol modified dNTPs.

9. The method according to any one of claims 1 to 8, wherein the first and second restriction enzymes are the same restriction enzyme.

10. 10. The process according to any one of claims 1 to 9, wherein step a) is carried out at a temperature below 50°C or the temperature is increased during the carrying out of step a).

11. The method of claim 10, wherein the temperature is increased from a starting ambient temperature.

12. 11. The method of claim 10, wherein the temperature is increased from a temperature in the range of 15 to 30°C to a temperature in the range of 40 to 50°C.

13. 13. The method according to any one of claims 1 to 12, wherein the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorometric dye, or a moiety enabling binding to a colorimetric or fluorometric dye, or an enzyme which generates a detectable signal after contact with a substrate.

14. 14. The method of claim 13, wherein the moiety capable of binding to a colorimetric or fluorometric dye is biotin.

15. 14. The method of claim 13, wherein the detectable signal is a colorimetric or fluorometric signal.

16. The method of any one of claims 1 to 15, wherein the detector species is detected by a change in an electrical signal.

17. The method according to any one of claims 1 to 16, wherein the portion of the second oligonucleotide probe that allows binding to a solid material is a single-stranded oligonucleotide, and the sequence of the single-stranded oligonucleotide portion may comprise three or more repeated copies of a 2-4 base DNA sequence motif.

18. The method according to any one of claims 1 to 17, wherein in step c) the presence of said detector species is detected by nucleic acid lateral flow.

19. 20. The method of claim 18, wherein the nucleic acid lateral flow utilizes one or more nucleic acids capable of sequence-specific hybridization with a moiety that enables binding of the second oligonucleotide probe to a solid material.

20. 20. The method of any one of claims 1 to 19, wherein step c) produces a colorimetric or electrochemical signal using carbon or gold.

21. 21. The method of any one of claims 1 to 20, wherein the first and / or second oligonucleotide primer comprises a stabilizing sequence upstream of the restriction enzyme recognition sequence and the cleavage site.

22. 22. The method of claim 21, wherein the stabilizing sequence is 5 or 6 bases in length.

23. The method according to any one of claims 1 to 22, wherein the hybridizing region of the first and / or second oligonucleotide primer is 9 to 16 bases in length.

24. 24. The method of any one of claims 1 to 23, wherein one of the first and second oligonucleotide primers is provided in excess of the other primer.

25. 25. The method of any one of claims 1 to 24, wherein the first and second hybridization sequences in the single-stranded target nucleic acid are separated by 0 to 15 bases.

26. 26. The method of claim 25, wherein the first and second hybridization sequences in the single-stranded target nucleic acid are separated by 3 to 15 bases.

27. 27. The method according to any one of claims 1 to 26, wherein in step b) the first or second single-stranded detection sequence of the at least one species in the amplification product comprises a sequence of at least 3 bases corresponding to 3 to 15 bases as defined in claim 26.

28. 28. The method of any one of claims 1 to 27, wherein the single-stranded target nucleic acid is single-stranded RNA, including single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA, or single-stranded DNA, including single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA, or single-stranded DNA obtained from double-stranded DNA by strand invasion, or single-stranded DNA obtained from double-stranded DNA by the use of a nuclease.

29. 30. The method of claim 28, wherein the single-stranded RNA is derived from double-stranded DNA or single-stranded DNA by use of reverse transcriptase.

30. 29. The method of claim 28, wherein the nuclease is a restriction endonuclease or exonuclease III.

31. The method of any one of claims 1 to 30, wherein the presence of two or more different single-stranded target nucleic acids of defined sequence is detected in the same sample.

32. the sample is a biological sample, and / or the single-stranded target nucleic acid is viral or derived from viral nucleic acid material, bacterial or derived from bacterial nucleic acid material, circulating cell-free DNA released from cancer cells or fetal cells, microRNA or derived from microRNA, and / or 32. The method of any one of claims 1 to 31, wherein detection of the single-stranded target nucleic acid is used to obtain data for the diagnosis, prognosis or monitoring of a disease or disease state; or detection of the single-stranded target nucleic acid is used to obtain data for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics.

33. 33. The method of claim 32, wherein the biological sample is a nasal or nasopharyngeal swab or aspirate, blood or a blood-derived sample, urine, a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample.

34. 33. The method of claim 32, wherein the disease is an infectious disease, the infectious disease being HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox.

35. 33. The method of claim 32, wherein the disease is cancer, and the cancer is colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma.

36. A kit for carrying out the method according to any one of claims 1 to 20, comprising: a) a first oligonucleotide primer and a second oligonucleotide primer, the first oligonucleotide primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to a first hybridization sequence in a single-stranded target nucleic acid of a defined sequence, and the second oligonucleotide primer comprising in a 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the single-stranded target nucleic acid; b) a first restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the first oligonucleotide primer and cleaving the cleavage site, and a second restriction enzyme which is not a nicking enzyme and is capable of recognizing the recognition sequence of the second oligonucleotide primer and cleaving the cleavage site; c) strand-displacing DNA polymerase; d) dNTPs, e) one or more modified dNTPs; f) a first oligonucleotide probe that is capable of hybridizing to and permitting detection of at least one species of a first single-stranded detection sequence in an amplification product produced in the presence of the single-stranded target nucleic acid; and g) a second oligonucleotide probe capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in the same strand of the at least one species in the amplification product, and attached to a solid material or a moiety that allows binding to the solid material; The kit, wherein at least one of the first and second oligonucleotide probes is blocked at the 3' end from extension by the strand-displacing DNA polymerase and cannot be cleaved by the first or second restriction enzyme.

37. 37. The kit of claim 36, wherein at least one of the first and second oligonucleotide probes is blocked at its 3' end from extension by the strand-displacing DNA polymerase and cannot be cleaved by the first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications to the restriction enzyme recognition sequence.

38. 38. The kit of claim 37, wherein the one or more modifications are phosphorothioate linkages.

39. The kit according to any one of claims 36 to 38, wherein one of the first and second oligonucleotide probes has 5 or more bases of complementarity with the hybridizing region of the first or second oligonucleotide primer or a reverse complement of the hybridizing region of the first or second oligonucleotide primer.

40. The kit of claim 39, wherein the first oligonucleotide probe has 5 or more bases of complementarity with a hybridizing region of one of the first and second oligonucleotide primers, and the second oligonucleotide probe has 5 or more bases of complementarity with a reverse complement of a hybridizing region of the other of the first and second oligonucleotide primers.

41. The kit of any one of claims 36 to 40, further comprising means for detecting the presence of a detector species produced in the presence of said single-stranded target nucleic acid.

42. The single-stranded target nucleic acid, the first oligonucleotide primer, and / or the second oligonucleotide primer, and / or the first restriction enzyme, and / or the second restriction enzyme, and / or the strand-displacing DNA polymerase, and / or the dNTPs, and / or the one or more modified dNTPs, and / or the first oligonucleotide probe, and / or the second oligonucleotide probe, and / or the first or second single-stranded detection sequence in the at least one species in the amplification product are defined as follows: at least one of the first and second oligonucleotide probes blocked at its 3' end from extension by the strand-displacing DNA polymerase comprises an additional region such that the 3' end of the at least one species in the amplification product to which the blocked oligonucleotide probe hybridizes can be extended by the strand-displacing DNA polymerase; The one or more modified dNTPs are alpha thiol modified dNTPs; the first and second restriction enzymes are the same restriction enzyme; the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorometric dye, or a moiety enabling binding to a colorimetric or fluorometric dye, or an enzyme that produces a detectable signal after contact with a substrate; the portion of the second oligonucleotide probe capable of binding to a solid material is a single-stranded oligonucleotide, the sequence of which may comprise three or more repeated copies of a 2-4 base DNA sequence motif; the first and / or second oligonucleotide primer comprises a stabilizing sequence upstream of the restriction enzyme recognition sequence and the cleavage site; the stabilizing sequence is 5 or 6 bases in length; the hybridizing region of the first and / or second oligonucleotide primer is 9 to 16 bases in length; providing one of said first and second oligonucleotide primers in excess of the other primer; the first and second hybridization sequences in the single stranded target nucleic acid are separated by 0 to 15 bases; the first and second hybridization sequences in the single stranded target nucleic acid are separated by 3 to 15 bases; the first or second single-stranded detection sequence of the at least one species in the amplification product comprises a sequence of at least 3 bases corresponding to the 3 to 15 bases separating the first and second hybridization sequences in the single-stranded target nucleic acid; the single-stranded target nucleic acid is single-stranded RNA, including single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA, or single-stranded DNA, including single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA, or single-stranded DNA derived from double-stranded DNA by strand invasion, or single-stranded DNA derived from double-stranded DNA by the use of a nuclease; the single stranded RNA is derived from double stranded DNA or single stranded DNA by use of reverse transcriptase; the nuclease is a restriction endonuclease or exonuclease III; The presence of two or more different single-stranded target nucleic acids of defined sequence is detected in the same sample; the sample is a biological sample; and / or the single-stranded target nucleic acid is viral or derived from viral nucleic acid material, bacterial or derived from bacterial nucleic acid material, circulating cell-free DNA released from cancer cells or fetal cells, microRNA or derived from microRNA; and / or detection of said single-stranded target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or disease state; or detection of said single-stranded target nucleic acid is used for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics; the biological sample is a nasal or nasopharyngeal swab or aspirate, blood or a blood-derived sample, urine, a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample; the disease is an infectious disease, the infectious disease being HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox; and / or The disease is cancer, and the cancer is colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma; The kit of any one of claims 36 to 41.

43. (A) a third oligonucleotide primer, the third oligonucleotide primer comprising, in the 5' to 3' direction, a recognition sequence and cleavage site for the first restriction enzyme and one strand of a region capable of hybridizing to the first hybridization sequence in the single-stranded target nucleic acid, and the third oligonucleotide primer being blocked at its 3' end from extension by the strand-displacing DNA polymerase; and / or (B) a fourth oligonucleotide primer, the fourth oligonucleotide primer comprising, in the 5' to 3' direction, a recognition sequence and cleavage site for the second restriction enzyme and one strand of a region capable of hybridizing with a reverse complementary strand of the second hybridization sequence in the single-stranded target nucleic acid, and the fourth oligonucleotide primer is blocked at its 3' end from extension by the strand-displacing DNA polymerase. The kit of any one of claims 36 to 42, further comprising:

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