NUCLEIC ACID DETECTION METHOD

Abstract

The present invention relates to methods for the detection of nucleic acids of defined sequence and to kits and devices for use in such methods; the methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.

Description

NUCLEIC ACID DETECTION METHOD TECHNICAL FIELD The present invention is directed to methods for the detection of nucleic acids of defined sequence and to kits and devices for use in such methods. BACKGROUND OF THE INVENTION Polymerase-based nucleic acid sequence amplification methods are widely used in the field of molecular diagnostics. The most established method, polymerase chain reaction (PCR), typically involves two primers for each target sequence and uses temperature cycling to achieve primer annealing, DNA polymerase extension, and denaturation of newly synthesized DNA in a cyclic exponential amplification procedure. The requirement for temperature cycling necessitates complex equipment that limits the use of PCR-based methods in certain applications. 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, and in Instead, it uses restriction enzymes combined with a strand displacement polymerase to separate the two strands of DNA. 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 nicked the restriction sites by by virtue of its ability to cleave only the unmodified strand of a hemiphosphorothioate form of its recognition site. A strand displacement polymerase extends to the 3' end of each nick and displaces the DNA strand in the 3' direction. Exponential amplification results from the coupling of sense and antisense reactions in which the displaced strands of a sense reaction serve as a target for an antisense reaction and vice versa. SDA typically takes about 1 hour to perform, which has greatly limited its potential for exploitation in the field of clinical diagnostics. Furthermore, the requirement for separate procedures for specific detection of the product after amplification and for starting the reaction add significant complexity to the method. Maples et al. (WO2009 / 012246) subsequently performed SDA using nicking enzymes, a subclass of restriction enzymes that are only capable of cleaving one of the two DNA strands after binding to their specific double-stranded recognition sequence. HE QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ referred to the method as the Nick and Extension Amplification Reaction (NEAR). NEAR, which employs nicking enzymes instead of restriction enzymes, has also been subsequently employed by others, who have attempted to improve the method using software-optimized primers (WO2014 / 164479) and through a hot start or controlled temperature reduction ( WO2018 / 002649). However, only a very small number of nicking enzymes are available and thus it is more challenging to find an enzyme with the desired properties for a particular application. A crucial disadvantage of SDA using either restriction enzymes or nicking enzymes (NEAR) is that it produces a double-stranded nucleic acid product and thus does not provide an intrinsic procedure for efficient detection of the amplification signal. This has significantly limited its usefulness in, for example, low-cost diagnostic devices. The double-stranded nature of the amplified product produced presents a challenge for coupling the amplification method to signal detection since it is not possible to perform hybridization-based detection without first separating the two strands. Therefore, more complex detection methods are required, such as molecular beacons and fluorophore / quencher probes, which can complicate assay protocols by requiring a separate procedural step and significantly reduce the potential to develop multiple assays. There is an important requirement for higher amplification methods of rapid, sensitive and specific detection of nucleic acid to overcome the limitations of SDA. The present invention relates to a method of amplification and detection of the target nucleic acid sequence that, in addition to a pair of primers with 5' restriction sites, uses additional oligonucleotide probes to produce a kind of detector that enables detection. signal efficiency. BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample comprising: a) Put the sample in contact with: Yo. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and a cleavage site and a region that is capable of hybridizing to a first hybridization sequence to the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ and cleavage site and a region that is capable of hybridizing to the reverse complement of a second hybridizing sequence downstream of the first hybridizing sequence in the target nucleic acid; ii. a strand displacement DNA polymerase; iii. the dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but is capable of annealing the recognition sequence of the first primer and cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; and I saw. a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product from step a) with: Yo. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is linked to a portion that allows its detection; and ii. a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in said at least one species within the amplification product and that is attached to a solid material or to a portion that allows attachment to a solid material; wherein hybridization of the first and second probes to said at least one species within the amplification product produces a detectable species; and c) detecting the presence of the detected species produced in step b) where QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ the presence of the species to be detected indicates the presence of the target nucleic acid in said sample. An embodiment of the method is illustrated in Figure 1. In various embodiments, in the presence of target nucleic acid, the method rapidly produces many copies of the detected species which is ideally suitable for sensitive detection. The present invention in several respects is advantageous over known methods in that it encompasses rapid amplification without temperature cycling as well as providing an intrinsic procedure for efficient detection of amplified product. The method of the invention overcomes a major disadvantage of SDA, including nicking enzyme SDA (NEAR), which is that SDA does not provide an intrinsic procedure for efficient detection of the amplification signal due to the double-stranded nature of the amplification product. amplification. The present method overcomes this limitation by using two additional oligonucleotide probes that hybridize to at least one species in the amplification product to facilitate its rapid and specific detection. The use of these two additional oligonucleotide probes, the first of which is attached to a portion that allows its detection and the second of which is attached to a solid material or a portion that allows its binding to a solid material, provides a number of additional advantages to the present invention over known methods such as SDA. For example, in embodiments of the invention where one of the oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase, it is not capable of being cleaved by the restriction enzyme(s) and is contacted with the sample simultaneously with the performance of step a), surprisingly no significant detrimental inhibition of amplification was observed and a pre-detector species containing a single-stranded region was produced efficiently. This aspect of the invention is counter-intuitive as it can be assumed that such a blocked probe could lead to asymmetric amplification that is strand-biased to the opposite amplification product to that comprised in the pre-detector species. In fact, such a pre-detector species is produced efficiently and is ideally suitable for efficient detection because the exposed single-stranded region is readily available for hybridization of the other oligonucleotide probe. The sample-intrinsic detection approach of the present method is in fundamental contrast to previous attempts to overcome this important limitation of SDA which involved performing asymmetric amplification, for example, by using unequal primer ratio with a goal of producing a excess of one amplicon strand over the other. The present method does not require asymmetric amplification nor does it have any requirement to produce an excess of one strand of the amplicon over the other and instead focuses on the production of the detectable species after hybridization of the first and second oligonucleotide probes. The focus of the QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ intrinsic sample detection of the present method involving the production of a detectable species is ideally suited for coupling with, among other detection methods, nucleic acid lateral flow, providing a simple, fast and inexpensive to perform the detection in step c), for example, by printing the second oligonucleotide probe on the lateral flow strip. When coupled to nucleic acid lateral flow the method also allows efficient multiplexing based on differential hybridization of multiple second oligonucleotide probes attached at discrete locations on the lateral flow strip, each with a different sequence designed for a different sequence. of target nucleic acid in the sample. In additional embodiments of the method, the efficiency of lateral flow detection is improved by using a single-stranded oligonucleotide as the portion within the second oligonucleotide probe that allows its binding to a solid material, and the reverse sequence complementary to said portion. is printed on the strip. The latter approach also allows the lateral flow strip to be optimized and fabricated as a single universal detection system across multiple target applications because the sequences attached to the lateral flow strip can be defined and do not need to correspond to the acid sequence. target nucleic(s). The integral requirement for the two additional oligonucleotide probes in the method of the invention thus provides many advantages over SDA, including SDA with nicking enzymes (NEAR). Since the present invention requires the use of restriction enzyme(s) that are not nicking enzymes and one or more modified dNTPs, it is fundamentally different for SDA to be performed using nicking enzymes (NEAR) and has many additional advantages over such dependent methods. of the nicking enzyme. For example, a much larger number of restriction enzymes that are not nicking enzymes are available compared to nicking enzymes, which means that the restriction enzyme(s) for use in the method of the invention they can be selected from a large number of potential enzymes to identify those with superior properties for a given application, eg reaction temperature, regulator compatibility, stability, and reaction rate (sensitivity). Due to this key advantage of the present method, we have been able to select restriction enzymes with a lower optimum temperature and faster rate than might be possible to achieve with nicking enzymes. Such restriction enzymes are much more convenient for exploitation in a low cost diagnostic device. Furthermore, the requirement for use of one or more modified dNTPs is an integral feature of the present invention that offers important advantages in addition to providing that restriction enzymes cleave only one strand of their restriction sites. For example, certain modified dNTPs, such as alpha thiol dNTPs, lead to a reduction in the melting temperature (Tm) of the DNA into which they are incorporated meaning that the oligonucleotide primers and probes in the method have a higher affinity for hybridization to the species QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ within the amplification product than any competitive complementary strand containing the modified dNTP produced during amplification. Furthermore, the reduction in Tm of the amplification product as a result of the modified dNTP base insertion facilitates separation of the double-stranded DNA species and thus improves the rate of amplification, reduces the optimum temperature and improves sensitivity. Alternatively, other modified dNTPs may increase the Tm of the DNA into which they are incorporated presenting more opportunities to design the method performance for a given application. Together with numerous advantages of the present invention over SDA, using either restriction enzymes or nicking enzymes (NEARs), they provide the utility of the method in low-cost, single-use diagnostic devices by virtue of the improved speed of simple amplification and visualization of the amplification signal which are not possible with known methods. Various embodiments of the aforementioned aspects of the invention, and additional aspects, are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic representation of the method according to one aspect of the invention. Figure 2. Schematic representation of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and contacted with the sample in step a). Figure 3. Schematic representation of steps b) and c) of the method where the portion that allows the binding of the second oligonucleotide probe to a solid material is a single-stranded oligonucleotide. Figure 4. Schematic representation of part of step a) of the method where the sample is additionally contacted with third and fourth oligonucleotide primers in step a). Figure 5. Embodiment of the method where the second oligonucleotide probe is attached to a solid material, a nitrocellulose lateral flow strip (see Example 1). Figures 6A and 6B. Carrying out the method where the first oligonucleotide probe is blocked at the 3' end of the extension by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and is brought into contact with the sample in step a) (see Example 2). Figure 7A, 7B, 7C and 7D. Realization of the method where the presence of two more QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ different target nucleic acids of defined sequence are detected in the same sample (see Example 3). Figure 8 . Embodiment of the method where the first and second hybridization sequences in the target nucleic acid are separated by 5 bases (see Example 4). Figure 9. Embodiment of the method where the portion that allows the 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 ). Figures 10A and 10B. Embodiment of the method wherein the portion that allows the binding of the second oligonucleotide probe to a 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 it is attached to a solid material (see Example 6). Figure 11. Use of the method for the detection of an RNA virus in clinical specimens (see Example 7). Figures 12A and 12B. Carrying out the method at different temperatures (see Example 8). Figures 13A and 13B. Embodiment of the method wherein the target nucleic acid is derived from double-stranded DNA by strand invasion (see Example 9). Figures 14A and 14B. Comparative realization of the method of the invention against known methods (see Example 10). DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for detecting the presence of a single-stranded target nucleic acid of 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 two strands in the sample such as by heat denaturation or through strand displacement activity of a polymerase, or derived from RNA for example. by the action of reverse transcriptase, or derived from double-stranded DNA eg by use of a nuclease, such as a restriction endonuclease or exonuclease III, or derived from an RNA / DNA hybrid eg via an enzyme such as Ribonudease H. The target nucleic acid may be single-stranded DNA derived from DNA in the sample by a DNA polymerase, helicase, or recombinase. Single-stranded sites within double-stranded DNA can be sufficiently exposed by hybridization and extension of the first oligonucleotide primer to initiate the method, for example by strand invasion where the transient opening of one or more base pairs of DNA within the QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ Double-stranded DNA occurs sufficiently to allow hybridization and extension of the 3' hydroxyl of the first oligonucleotide primer, or by spontaneous opening of DNA base pairs, transient conversion to Hoogsteen pairs, or productive nicking of DNA by enzyme approaches. restriction or thermochemical. 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 such as by heat denaturation, or single-stranded RNA derived from double-stranded DNA for example by transcription. The method involves in step a) contacting the sample with: (i) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a recognition sequence of restriction enzyme and cleavage site and a region that is capable of hybridizing to a first hybridization sequence in the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence. restriction enzyme and cleavage site and a region that is capable of hybridizing to the reverse complement of a second hybridizing sequence 5' of the first hybridizing sequence in the target nucleic acid; (ii) a strand displacement 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 primer and of cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded , cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by 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 second primer recognition sequence and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs. When the target nucleic acid to be detected in the sample is double-stranded either strand can be considered the single-stranded target nucleic acid of the method since one of the two oligonucleotide primers is capable of hybridization to one strand and the other oligonucleotide primer is capable of hybridization. hybridization to the other strand. Typically, the oligonucleotide primers used in the method are DNA primers that form with the target DNA or RNA a double-stranded DNA or a hybrid duplex comprising both RNA and DNA strands. However, primers comprising other nucleic acids, such as non-natural bases and / or alternative backbones, may also be used. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ In the presence of the target nucleic acid the first oligonucleotide primer hybridizes to the first hybridizing sequence in the target nucleic acid. After such annealing, the 3' hydroxyl group of the first primer is extended by strand displacement DNA polymerase or, optionally, in the case of an RNA target nucleic acid a reverse transcriptase (eg M-MuLV). , to produce a double-stranded species containing the extended first primer and the target nucleic acid (see Figure 1). Strand displacement DNA polymerase or, when present, reverse transcriptase uses the dNTPs and the one or more modified dNTPs in said extension. The one strand of a restriction enzyme recognition sequence and cleavage site at the 5' end of the first primer typically does not anneal since the reverse complementary sequence thereto is generally not present in the target nucleic acid sequence. Thus the first primer is generally used to introduce said one strand of a restriction enzyme recognition sequence and cleavage site into the subsequent amplification product species. After first primer extension, target deletion occurs. Target deletion makes it possible for the extended first primer species to anneal to the second oligonucleotide primer to the reverse complement of the second annealing sequence. When the target nucleic acid is RNA, target removal can be achieved, for example, by RNase H degradation of the RNA, achieved through the RNase H activity of reverse transcriptase if present or through the separate addition of this. enzyme. Alternatively, when the target nucleic acid is single-stranded DNA, including a single-stranded region within the double-stranded DNA can be achieved by strand displacement using an additional downstream primer or skip primer. Alternatively, such target removal may occur after spontaneous dissociation, particularly if only a short extension product of a given target nucleic acid molecule has been produced, or it may occur via strand invasion where the transient opening of one or more DNA base pairs within the first double-stranded extended primer species occurs sufficiently to allow annealing and extension of the 3' hydroxyl of the second oligonucleotide primer with strand displacement. After hybridization of the second oligonucleotide primer to the reverse complement of the second hybridization sequence, the strand displacement DNA polymerase extends the 3' hydroxyl of said primer using the dNTPs and the one or more modified dNTPs. The 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 complementary strand that act to block cleavage of said strand by said first restriction enzyme. The first restriction enzyme recognizes its recognition sequence and cleaves only the first primer strand from the cleavage site, creating a 3' hydroxyl that is extended by strand displacement DNA polymerase using the dNTPs and the one or more QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ dNTPs modified and displacing the first primer strand. The double-stranded restriction recognition sequence and cleavage site for the second restriction enzyme is formed with one or more dNTP-modified bases incorporated into the reverse complementary strand that act to block cleavage of said strand by said second restriction enzyme. Thus a double-stranded species is produced in which the two primer sequences are juxtaposed and the partially blocked restriction site of the first restriction enzyme and the second restriction enzyme is present. Cleavage by the first restriction enzyme of the first primer strand and by the second restriction enzyme of the second primer strand then occurs, and two double-stranded species are produced, one comprising the first primer sequence and the other comprising a second primer sequence. Sequential excision and displacement of the first primer strand and second primer strand then occurs in a cyclic amplification procedure wherein the displaced first primer strand acts as a target for the second primer and the displaced second primer strand acts as a target. a target for the first primer. In the presence of target nucleic acid, the amplification product is produced without any requirement for temperature cycling. An integral aspect of the present invention is that rather than directing the detection of the amplification product from step a), a detector species is produced after specific hybridization of both a first and a second oligonudeotide probe to at least one species. within the amplification product. The first oligonudeotide probe, which binds to a portion allowing its detection, hybridizes to a first single-stranded detection sequence in said at least one species. The second oligonudeotide probe, which binds to a solid material or a portion that allows it to bind to a solid material, hybridizes to a second single-stranded detection sequence 5' or 3' of the first single-stranded detection sequence in said at least one species. It will be apparent to a skilled person, with reference to Figure 1, that the amplification product comprises a number of different species, such as species comprising single-stranded detection sequences, consisting of the complete or pardal sequence or the reverse complementary sequence. of both the first and second primers, said sequences may be separated by the sequence derived from the target in the event that the first and second primer-binding hybridization sequences in the target nucleic acid are separated by one or more bases. It will further be apparent that any such species can be selected to hybridize to the first and second oligonudeotide probes to form the detector species. The detector species produced in step b) is detected in step c), wherein the presence of the detector species indicates the presence of the target nucleic acid in the sample. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ By using two oligonucleotide probes, one for detection and one for binding to a solid material, the method of the invention provides rapid and efficient detection of the signal, which exceeds the requirement of more complex secondary detection methods and provides efficient visualization of the signal produced in the presence of the target, such as by lateral flow of nucleic acid. The method of the invention may be performed wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by strand displacement DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes. Thus, according to an additional 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) Put the sample in contact with: Yo. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and a cleavage site and a region that is capable of hybridizing to a first hybridization sequence to the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of reverse complement hybridization of a second hybridizing sequence downstream of the first hybridizing sequence in the target nucleic acid; ii. a strand displacement DNA polymerase; iii. the dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but is capable of annealing the recognition sequence of the first primer and cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; and I saw. a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the chain QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ reverse complementary is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product from step a) with: Yo. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is linked to a portion that allows its detection; and ii. a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in said at least one species within the amplification product and that is attached to a solid material or to a portion that allows attachment to a solid material; wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, and where hybridization of the first and second probes to said at least one species within the amplification product produces a detectable species; and c) detecting the presence of the detect species produced in step b) wherein the presence of the detect species indicates the presence of the target nucleic acid in said sample. In one said embodiment a blocked oligonucleotide probe is rendered incapable of being cleaved by either the first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications such as a phosphorothioate bond. In a further embodiment, a blocked oligonucleotide probe is brought into contact with the sample simultaneously for the performance of step a), that is to say during the performance of step a), so that it is present during the production of the amplification product in the presence of the target nucleic acid. Thus, according to an additional 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) Put the sample in contact with: Yo. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ a restriction enzyme recognition sequence and a cleavage site and a region that is capable of hybridizing to a first hybridization sequence in the target nucleic acid, and said second primer comprises in the 5-direction ' to 3' a strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridizing to the reverse complement of a second hybridizing sequence upstream of the first hybridizing sequence in acid target nucleic; ii. a strand displacement DNA polymerase; iii. the dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but is capable of annealing the recognition sequence of the first primer and cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; and I saw. a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product from step a) with: Yo. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is linked to a portion that allows its detection; and i. a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in said at least one species within the amplification product and that is attached to a solid material QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ or to a portion that allows it to be attached to a solid material; wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase, is not capable of being cleaved by either of the first or second restriction enzymes and is brought into contact with the sample simultaneously for carrying out step a), and where the hybridization of the first and second probes to said at least one species within the amplification product produces a detectable species; and c) detecting the presence of the detect species produced in step b) wherein the presence of the detect species indicates the presence of the target nucleic acid in said sample. For example, in the embodiment illustrated in Figure 2, the first oligonucleotide probe is blocked and hybridizes to the first single-stranded detection sequence in at least one species within the amplification product to form a pre-detector species containing a single-stranded region. Said at least one species can be extended by the strand displacement DNA polymerase by extending its 3' hydroxyl group and thereby further establishing said pre-detector species. Thus, in said embodiment the blocked oligonucleotide probe comprises an additional region so that the 3' end of the species within the amplification product to which the blocked oligonucleotide probe hybridizes can be extended by strand displacement DNA polymerase. . A Stabilized Pre-detector Species is produced as shown in Figure 2. The skilled person will appreciate that this additional pre-detector species stabilization region in the blocked oligonucleotide probe will be 5' of the region that hybridizes to. to either the first or second single-stranded detection sequence in the at least one species within the amplification product. In embodiments using a blocked oligonucleotide probe the hybridization sequence of the blocked oligonucleotide probe and the relevant concentrations of primers can be optimized so that a certain proportion of the relevant species produced in the amplification product hybridizes to the blocked oligonucleotide probe. oligonucleotide blocked in each cycle and the remaining species of such species remain available to participate in the cyclical amplification procedure. The oligonucleotide probe is blocked from extension, for example, by the use of a 3' phosphate modification and, in this embodiment, also binds to a moiety that allows its detection, such as a 5' biotin modification . Alternatively, a single 3' modification can be used to block the extension and as a portion allowing its detection. Various other modifications are available to block the 3' end of oligonucleotides such as a C-3 spacer; alternatively, mismatched base(s) may be used. Such a pre-detector species is ideally suitable for efficient detection because the exposed single-stranded region remains readily available for hybridization to the second oligonucleotide probe. The QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ second oligonucleotide probe can be attached to the nitrocellulose surface of a lateral flow strip of nucleic acid so that when the pre-detector species flows over it, sequence-specific hybridization readily occurs and species detected is located at a defined location on the strip. A dye that binds to the detection portion, such as a streptavidin-bound carbon, gold, or polystyrene particle, which may be present on the conjugate pad of the nucleic acid lateral flow strip or during the amplification reaction, provides a rapid color-based display of the presence of the detectable species produced in the presence of the target nucleic acid. In another embodiment it is the second oligonucleotide probe that has extension blocked at the 3' end by strand displacement DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes and is contacted with the sample simultaneously to the performance of step a). The second oligonucleotide probe may be attached to a solid material, such as the surface of an electrochemical probe, 96-well plate, bead, or array surface, prior to being brought into contact with the sample, or it may be attached to a portion that allows its union to a solid material. A certain proportion of at least one species produced during amplification anneals to the second oligonucleotide probe after its production, rather than annealing to the relevant reaction primer to further participate in the cyclic amplification process. After hybridization to the second oligonucleotide probe, said species are extended by the polymerase over the oligonucleotide probe to produce the stabilized pre-detector species. The first oligonucleotide probe and the detection portion can also be brought into contact with the sample simultaneously with the performance of step a) and could be located on said surface at the site of the second oligonucleotide probe. By detecting the accumulation of the detection moiety at the site during the amplification procedure a real time signal could be obtained providing a quantification of the number of copies of the target nucleic acid present in the sample. Thus according to one embodiment of the invention, two or more of steps a), b) and c) are performed simultaneously. In carrying out those embodiments where one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes and is contacted with the sample simultaneously to the performance of step a), we have not observed any significant inhibition of the amplification rate, indicating that the pre-detector species accumulates in real time without altering the optimal cyclical amplification procedure. This is contrary to attempts to design asymmetric SDA by using unequal primer ratio with the goal of producing an excess of one amplicon strand over the other. Rather than looking to use the locked oligonucleotide probe to remove an amplicon strand from the reaction and increase QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ Thus the ratio of the other strand, the present invention focuses on the production and detection of the detecting species that exploits a locked probe to facilitate exposure of a single-stranded region during the amplification procedure. Thus, not only do we not observe any inhibitory effect in the amplification procedure in said modalities, but we also observe a surprising improvement in the signal produced corresponding to an increase in the amount of the species detected, of at least 100 times in certain modalities, see Example 2 (Figures 6A-6B). In addition, said embodiments of the method of the invention wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second oligonucleotide enzymes. restriction and is contacted with the sample simultaneously to the performance of step a), represents a fundamental advantage over the reported attempts to integrate the NEAR with the lateral flow of nucleic acid in a multistep procedure without blocked probes. For example, in WO2014 / 164479 a long incubation of 30 minutes at 48°C was required to visualize the amplification product using nucleic acid lateral flow, which represents a major impediment to the use of that method in a diagnostic device of point of care, particularly a low-cost or single-use device. In stark contrast, the method of the invention easily performs equivalent amplification in under 5 minutes and at a lower incubation temperature, eg 40-45°C. In a further head-to-head comparative study (see Example 10), the method of the invention demonstrates a surprisingly superior speed compared to the prior art method (WO2014 / 164479) resulting from a combination of the use of a restriction enzyme that it is not a nicking enzyme, the use of a modified dNTP base and the use of said blocked oligonucleotide probe. It will also be appreciated that the other of the first and second oligonucleotide probes may be blocked at the 3' end of the extension by DNA polymerase, and / or is not capable of being cleaved by either the first or second restriction enzymes. , as described before. An integral aspect of the method is the use of one or more restriction enzyme that is not a nicking enzyme, but is capable of recognizing its recognition sequence and cleaving only one strand from its cleavage site when said recognition sequence and cleavage site cleavage are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into the reverse complementary strand by a strand displacement DNA polymerase using one or more modified dNTPs, e.g. a dNTP that confers nuclease resistance after its incorporation by a polymerase. A restriction enzyme [or restriction endonuclease] is a broad class of enzyme that cleaves one or more phosphodiester bonds on one or both strands of a double-stranded nucleic acid molecule at specific cleavage sites after binding to a restriction sequence. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ specific recognition. A large number of restriction enzymes are available, with over 3,000 reported and over 600 commercially available, covering a wide range of different physicochemical properties and recognition sequence specificities. A nicking enzyme [or nicking endonuclease] is a particular subclass of restriction enzyme, which is only capable of cleaving one strand of a double-stranded nucleic acid molecule at a specific cleavage ratio after binding to a specific recognition sequence, leaving the other string intact. Only a very small number (c.10) of nicking enzymes are available including both natural and engineered enzymes. Notching enzymes include the strand cleavers Nb.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.BssSI and Nb.BtsI and the upper strand cleavers Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt .BstNBI and Nt.CviPII. Restriction enzymes that are not nicking enzymes, which are used exclusively in the method of the invention, despite being capable of cleaving both strands of a double-stranded nucleic acid, can also under certain circumstances cleave or nicked only one strand at their site. double-stranded DNA cleavage after binding to its recognition sequence. This can be done in several ways. Of particular relevance to the present method, this can be achieved when one of the strands within the double-stranded nucleic acid at the cleavage site becomes incapable of being cleaved because a strand from the target site of the double-stranded nucleic acid is modified such that the phosphodiester bond of the cleavage site on one of the strands is protected using a nuclease resistant modification, such as a phosphorothioate (PTO), boranophosphate, methylphosphate, or peptide internucleotide bond. Certain modified internucleotide linkages, eg PTO linkages, can be chemically synthesized within oligonucleotide probes and primers or integrated into a double-stranded nucleic acid by a polymerase, such as by use of one or more alpha-thiol-modified deoxynucleotides. Thus, in one embodiment the one or more modified dNTPs is a modified alpha thiol dNTP. Typically the S isomer is employed which incorporates and confers nuclease resistance more efficiently. Due to the very large number of restriction enzymes available that are not nicking enzymes, a wide range of enzymes with different properties are available to be examined for desired performance characteristics, eg temperature profile, rate, regulator compatibility, cross compatibility. of polymerase, recognition sequence, thermostability, manufacturability etc., for use in the method for a given application. In contrast, the fact that only a small number of nicking enzymes are available limits the potential of prior art methods using nicking enzymes, and can lead to lower reaction rates (sensitivity, time to results) and higher temperature. reaction, for example. Restriction enzymes that are not nicking enzymes QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ selected for use in the method may be natural or engineered enzymes. In selecting the restriction enzyme that is not a nicking enzyme for use in the method the skilled person will recognize that it is necessary to identify an enzyme with an appropriate cleavage site to ensure that a modification is incorporated at the correct position to block cleavage. from the relevant chain and not from another chain. For example, in an embodiment where a modified dNTP is used, such as an alpha thiol dNTP, it may be preferable to select a restriction enzyme with a cleavage site that falls outside of the recognition sequence, such as a restriction enzyme asymmetric with a non-palindromic recognition sequence, to provide sufficient flexibility to position the primers so that the target nucleic acid sequence contains the modified nucleotide base at the appropriate location to block excision of the relevant strand after its incorporation. For example, if the alpha thiol dATP is used, the reverse complementary sequence of the restriction enzyme cleavage site in the relevant oligonucleotide primer could contain an Adenosine base downstream of the cleavage position on said reverse complementary strand but not contain an Adenosine base downstream of the cleavage site in the primer sequence, to ensure that the primer is properly cleaved in performing the method. Therefore asymmetric restriction enzymes with a non-palindromic recognition sequence that cleave outside of their recognition sequence are ideally suitable for use in the present invention. Partial or degenerate palindromic sequence recognition restriction enzymes that cleave within their recognition site may also be used. Nuclease-resistant nucleotide linkage modifications, for example PTO, can be used to block cleavage of any strand by a wide range of commercially available double-stranded cleavage agents of several different classes, including US-type and IIG-type restriction enzymes. with both partial or degenerate palindromic and asymmetric restriction recognition sequences, to enable their use in the method of the invention. The restriction enzyme(s) are typically employed in the method in an amount of 0.1 - 100 Units, where one unit is defined as the amount of agent required to digest 1 pg of T7 DNA in 1 hour at a given temperature. (for example 37°C) in a total reaction volume of 50 μΙ. However, the amount depends on a number of factors such as the activity of the selected enzyme, the concentration and form of the enzyme, the anticipated concentration of the target nucleic acid, the volume of the reaction, the concentration of the primers, and the temperature. of reaction, and should not be considered limiting in any way. Those skilled in the art will understand that a restriction enzyme employed in the method will require a buffer and suitable salts, eg, divalent metal ions, for effective and efficient function, pH control, and stabilization of the enzyme. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ 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 a single enzyme that is compatible with other reaction components needs to be identified, optimized for method performance, manufactured, and stabilized. The use of a single restriction enzyme also simplifies the design of oligonucleotide primers and supports symmetry of the amplification procedure. In the method the restriction enzymes cleave only one strand of the duplex nucleic acid, and thus upon cleavage present an exposed 3' hydroxyl group which can act as an efficient priming site for a polymerase. A polymerase is an enzyme that synthesizes nucleic acid strands or polymers by extending a primer and generating a reverse complementary copy of a DNA or RNA template strand using base-pairing interactions. A polymerase with strand displacement capability is employed in carrying out the method so that strands are appropriately displaced to affect the amplification procedure. The term "strand displacement" refers to the ability of a polymerase to displace DNA encountered during synthesis in the 3' direction. A range of commercially available strand displacement polymerases operating at different temperatures have been characterized. For example, the Ph¡29 polymerase has a very strong ability to displace strands. Bacillus species polymerases, such as Bst DNA Polymerase Long Fragment, typically exhibit high strand displacement activity and are very convenient for use in carrying out the method. The E. co / / Klenow (exo-) fragment is another widely used strand displacement polymerase. Strand displacement polymerases can be easily designed, such as KlenTaq such as by cloning only the polymerase active domain of an endogenous enzyme and inactivating any exonuclease activity. In order to carry out the method in which the single-stranded target nucleic acid is RNA, the activity of RNA-dependent DNA synthesis (reverse transenptase) is also required. Said activity can be carried out by strand displacement polymerase and / or by a additional reverse transcriptase enzyme separated in step a), eg MMuLV or AMV. The polymerase(s) are typically employed in the relevant steps of the method in an appropriate amount that is optimized depending on the enzyme, the concentration of reagents, and the desired temperature of the reaction. For example, 0.1-100 Units of a Bacillus polymerase can be used, where one unit is defined as the amount of enzyme that will incorporate 25 nmoles of dNTPs into acid insoluble material in 30 minutes at 65°C. However, the amount depends on a number of factors such as the activity of the polymerase, its concentration and shape, the anticipated concentration of the target nucleic acid, the volume of the reaction, the number and QbRnnn / ιζηζ / Β / γίΛΐ concentration of oligonucleotide primers and reaction temperature, and is not to be considered limiting in any way. Those skilled in the art will know that polymerases require dNTP monomers to have polymerase activity and also that they require an appropriate buffer, with components such as buffer salts, divalent ions, and stabilizing agents. In addition, one or more modified dNTPs are used in the method to block reverse complementary strand cleavage from the primers after incorporation by strand displacement polymerase. Typically when using a single modified dNTP, the dNTPs used in the method must omit the corresponding base. For example, in an embodiment where the modified dNTP is alpha thiol dATP, the dNTPs should comprise only dTTP, dCTP, and dGTP and should not include dATP. Deletion of the corresponding native dNTP base ensures that all required downstream cleavage sites within the reverse complementary sequence of the primers are blocked because only the modified base is available for incorporation by the polymerase, however it is not essential. complete or partial removal of the corresponding native dNTP base. dNTPs can typically be used in the method at concentrations similar to those employed in other polymerase methods, such as concentrations ranging from 10 micromolar to 1 millimolar, although the dNTP concentration for the method can be optimized for any given enzyme and reagents. , to maximize activity and minimize ab initio synthesis to avoid generation of background signal. Since certain polymerases may exhibit a lower rate of incorporation with one or more bases than modified dNTPs, the one or more modified bases can be used in the method at a higher relative concentration than unmodified dNTPs, such as five times the concentration. higher, although this should be considered non-limiting. The use of one or more modified dNTPs is an integral feature of the present invention that offers an important advantage in addition to providing that restriction enzymes cleave only one strand of their restriction sites. For example, certain modified dNTPs, such as alpha thiol dNTPs, lead to a reduction in the melting temperature (Tm) of the DNA into which they are incorporated meaning that the oligonucleotide primers and probes in the method have a higher affinity. for hybridization to the species within the amplification product than any competitive antisense strand containing the modified dNTP produced during amplification. This key feature increases the rate of amplification because, for example, when one of the displaced strands anneals to its reverse complement to produce a nonproductive endpoint species, it dissociates more easily than productive annealing of that displaced strand to an additional primer. due to the presence of one or more modified bases leading to a reduction in the hybridization Tm. It has been reported that internucleotide phosphorothioate linkages can reduce the Tm, the temperature at which exactly one half of QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ the single chains of a duplex, by 1-3°C per addition, a substantial change in physicochemical properties. We have also observed an increased rate of strand displacement when phosphorothioate nucleotide bonds are present in a DNA sequence. Furthermore, the oligonucleotide probes used in the method, whether contacted with the sample simultaneously with the performance of step a) or subsequently, possess a higher affinity for those species within the amplification product than any competitive modified species and thus they may preferentially hybridize or even displace hybridized strands to facilitate production of the detectable species. The reduced Tm and increased species displacement of the amplification product as a result of the modified internucleotide linkages it contains serves to fundamentally increase the speed of the method and reduce the temperature required for rapid amplification to occur. In addition to the increased speed that results from the use of one or more modified nucleotides, the hybridization specificity of the method's oligonucleotide primers and probes is also increased. Since typically all the bases of a particular nucleotide are substituted within the amplification product, the hybridization sites of primers and probes typically contain modified bases, and the reduced Tm resulting from internucleotide phosphorothioate bonds, for example, means that they are likely to be sequence mismatches by non-specific hybridization are less tolerated. Thus the comprehensive feature of the method of the invention for one or more modified dNTPs leads to fundamental benefits that increase both the sensitivity and the specificity of amplification and are in stark contrast to known methods without such a requirement for modified nucleotides, such as NEAR (WO2009 / 012246), including NEAR variants with software-optimized primers (WO2014 / 164479) or a hot start or temperature controlled turndown (WO2018 / 002649). There are a number of different modified dNTPs, such as modified dNTPs that confer nuclease resistance after incorporation by a polymerase, and can be used in the method to achieve resistance to restriction enzyme cleavage and, in embodiments, other features to increase the performance of the method for a given application. In addition to alpha thiol dNTPs that provide nuclease resistance and a reduction in Tm, modified dNTPs that are reported to have the potential for polymerase incorporation and to confer nuclease resistance include nucleotide equivalent derivatives, such as Borane derivatives, modified 2'-O-Methyl (2'OMe) bases and 2'-Fluoro bases. Other modified dNTPs or equivalent compounds that can be incorporated by polymerases and used in method embodiments to increase particular method properties include those that decrease binding affinity, for example Inosine-5'-Tr¡phosphate or 2'- Deoxyzebular to-5'-Tr¡phosphate, those that QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ increase the specificity of the binding, for example 5-Methyl-2'-deoxycytidine-5'-Triphosphate or 5-[(3Indolyl)propionam¡da-N-al¡l]-2' -deoxi¡ur¡d¡na-5'-Trifosfato, and those that increase the synthesis of the rich regions in GC, for example 7-deaza-dGTP. Certain modifications can increase the Tm providing additional potential for monitoring hybridization events in method modalities. 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 relevant polymerase and restriction enzymes and the melting temperature of the annealing regions of the oligonucleotide primers. Notably the method does not use temperature cycling in step a). Furthermore, amplification step a) does not require any controlled temperature swing, nor any hot or warm start, pre-heating or controlled temperature decrease. The method allows the steps to be carried out over a wide temperature range, for example 15°C to 60°C, such as 20 to 60°C, or 15 to 45°C. According to one embodiment, step a) is performed at a temperature of not more than 50°C, or around 50°C. Given the wide range of restriction enzymes other than nicking enzymes available for use in the method, it is possible to select for restriction enzymes with a fast rate at relatively low temperatures compared to alternative methods using nicking enzymes. The use of one or more modified nucleotides also reduces the required amplification temperature. In addition to having the potential for a lower optimum temperature profile compared to known methods, the method of the invention can be performed over an unusually wide range of temperatures. Such features are highly attractive for use of the method in a low-cost diagnostic device, where controlled heating imposes complex physical constraints that increase the cost of goods for such a device to a point where a single-use or instrument-free device it is not commercially viable. A number of tests have been performed using the method that can perform rapid detection of target nucleic acid at room temperature or near 37°C, for example. As such, in a further embodiment step a) is performed at a temperature of not more than 45°C, or near 45°C. It may be preferable to start the method at a temperature lower than the target temperature to simplify user steps and decrease overall time to result. As such in a further embodiment of the method, the temperature of step a) is increased during amplification. For example, the temperature of the method can start at room temperature, such as 20°C, and increase over a period, such as two minutes, to the final temperature, such as about 45°C or 50°C. In one embodiment the temperature is increased during the performance of step a), such as an increase from an initial ambient temperature, for example in the range of 15-30°C, up to a temperature in the range of 40-50°C. . The low temperature potential and versatility of the method of the invention means QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ which, in contrast to known methods, is compatible with the conditions required for a range of other assays, such as immunoassays or enzymatic assays for the detection of other biomarkers, such as proteins or small molecules. Thus the method can be used, for example, for the simultaneous detection of both nucleic acids and proteins or small molecules of interest within a sample. The components required to perform the method, including the restriction enzymes that are not nicking enzymes, strand displacement DNA polymerase, oligonucleotide primers, oligonucleotide probes, the dNTPs, and one or more modified dNTPs, can be lyophilized or dried. by freezing for stable storage and the reaction can then be activated by rehydration, such as after addition of the sample. Such lyophilization or freeze-drying for stable storage typically requires the addition of one or more excipients, such as trehalose, prior to drying of the components. A very wide range of such excipients and stabilizers for lyophilization or freeze drying are known and are available for testing to identify a suitable composition for the components required for method performance. It will be apparent to a person skilled in the art that the method of the invention, which is a polymerase-based amplification method, can be improved by the addition of one or more additives that have been shown to improve PCR or other polymerase-based amplification methods. of polymerase. Such additives include but are not limited to tetrahydrothiophene 1-oxide, L-lysine free base, Larginine, glycine, histidine, 5-aminovaleric acid, 1,5-diamino-2-methylpentane, N,N'diisopropylethylenediamine, tetramethylenediamine (TEMED), tetramethylammonium chloride, tetramethylammonium oxylate, methyl sultanacetamide, hexadecyltrimethylammonium bromide, betaine aldehyde, tetraethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, tetrabutylammonium chloride, tetrapropylammonium chloride, formamide , dimethylformamide (DMF), N-methylformamide, Nmethylacetamide, Ν,Ν-dimethylacetamide, L-threonine, Ν,Ν-dimethylethylenediamine, 2-pyrrolidone, HEP (Nhydroxyethylpyrrolidone), NMP (N-methylpyrrolidone) and 1-methyl, l- Cyclohexyl-2-pyrrolidone (pyrrolidinones), δ-valerolactam, N-methylsuccinimide, 1-formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose, glycerol, Tween-20, DMSO, betaine, and BSA. Our investigations have revealed that the present method is effective over a wide range of target nucleic acid levels including low to very low detection, including single copy numbers. Oligonucleotide primers are typically provided in vast excess over the target nucleic acid. Typically the concentration of each primer is in the range 10 to 200 nM although that should be considered non-limiting. A higher primer concentration can increase the hybridization efficiency and therefore increase the speed of the reaction. However, non-specific background effects, such as primer-dimers, can also be QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ observing at high concentration and therefore the concentration of the first and second oligonucleotide primers is part of the optimization procedure for any given run using the method. In one embodiment the first and second oligonucleotide primers are provided at the same concentration. In an alternative embodiment one of the first and second oligonucleotide primers is provided in excess of the other. The reaction rate can be reduced in modalities where one of the primers is supplied in excess of the other due to the natural symmetry of the cyclic amplification procedure, however in certain circumstances it can be used to reduce the non-specific background signal in the cyclic amplification process. method and / or to increase the ability of the first and second oligonucleotide probes to hybridize to produce the detector species. It is desirable that both primers be present at such a level as not to become limiting before sufficient detector species have been produced for detection with the selected detection medium. There are a number of considerations for the design of the oligonucleotide primers for carrying out the method. Each of the first and second oligonucleotide primers should comprise in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and cleavage site and a hybridization region, wherein said hybridization region is capable of hybridizing. to a first hybridizing region in the target nucleic acid in the case of the first primer and to the reverse complement of a second hybridizing sequence upstream of the first hybridizing sequence in the target nucleic acid in the case of the second primer. Thus a primer pair is designed to amplify a region of the target nucleic acid. The restriction enzyme recognition sequence of the primers is typically not present within the target nucleic acid sequence and thus forms a pendant during initial hybridization events before being introduced into the amplicon (see Figure 1). In the case where an asymmetric restriction enzyme is used the cleavage site is typically upstream of the recognition sequence and may therefore, optionally, be present within the annealing sequence of the primer. Oligonucleotide primers are designed so that after cleavage in the method, the sequence 5' of the cleavage site forms an upstream primer with sufficient melting temperature (Tm) to remain hybridized to its reverse complementary strand under the conditions of the oligonucleotides. desired reaction conditions and to displace the strand downstream of the cleavage site after extension of the 3' hydroxyl group by the strand-displacing DNA polymerase. Thus an additional stabilization region can be included at the 5' end of the oligonucleotide primers, the optimal length of which is determined by the position of the cleavage site with respect to the recognition sequence for the relevant restriction enzyme and other factors such as as the temperature to be used for amplification in step a). Thus in one embodiment the first and / or second oligonucleotide primers comprise a sequence QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ stabilizer upstream of the restriction enzyme recognition sequence and cleavage site, such as at the 5' end, and eg 5 or 6 bases in length. During primer design it is necessary to define the sequence and length of each hybridization region to allow for optimal sequence-specific hybridization and strand displacement to ensure specific and sensitive amplification in the method. The positioning of the primers within the target nucleic acid to be detected, for example within the genome of a viral or bacterial pathogen, can be varied to define the sequence of the hybridizing region of the primers and thus select primers with the sensitivity and optimal specificities for amplification and compatibility with oligonucleotide probes. Therefore, different pairs of primers can be studied to identify the optimal sequence and positioning for carrying out the method. Typically the length of the annealing region of the primers is designed such that their theoretical Tm allows efficient annealing at the desired reaction temperature but is also easily displaced after cleavage. During primer design, the theoretical Tm of the hybridizing sequence and the sequence of the displaced strands are considered in the context of the likely temperature of the reaction and the selected restriction enzyme, which is balanced by the theoretical improvement to the sequence-derived binding specificity that can result as sequence length increases. Our various investigations have indicated considerable versatility in the design of the primers to be used effectively in the method. In one embodiment the hybridizing region of the first and / or second oligonucleotide primers is between 6 and 30, eg 9 and 16, bases in length. In other embodiments, modifications, such as non-natural bases and alternative internucleotide linkages or abasic sites, may be employed in the hybridizing regions of the primers to refine their properties and method performance for a particular application. For example a modification that increases Tm, such as PNA, LNA or G-clamp may allow for a shorter and more specific primer annealing region enabling a shorter amplicon and thus increasing amplification rate. Our various investigations have revealed that the speed of the method and its sensitivity can be increased by having a short amplicon and thus in certain modalities it may be preferable to trim both the full length of the primers, including their annealing sequence, as well as to position the primers with only a short gap, such as 10 or 15 nucleotide bases or less, between the first and second hybridizing sequences in the target nucleic acid. In one embodiment the first and second hybridizing sequences in the target nucleic acid are separated by 0 to 15 or 0 to 6 bases, in certain embodiments they are separated by 3 to 15 or 3 to 6 bases, for example 5, 7 or 11 bases . In a further embodiment the hybridization sequences overlap, such as by 1 to 2 bases. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ There are a number of considerations in designing the sequence of oligonucleotide probes for use in the method. First, the region in the first oligonucleotide probe that hybridizes to the first single-stranded detection sequence and the region in the second oligonucleotide probe that hybridizes to the second single-stranded detection sequence are typically designed so that they cannot be interchanged. overlap or have minimal overlap, to allow both oligonucleotide probes to bind to the at least one species within the amplification product at the same time. They are also typically designed to hybridize primarily to the sequence that falls between the cleavage site position on one strand of the amplification product species and the position opposite the cleavage site on the reverse complementary strand thereto to ensure that the one or more species within the amplification product are targeted efficiently and that both oligonucleotide probes bind to the same strand. For any given primer pair, any strand can be selected for targeting by the oligonucleotide probes. Since oligonucleotide probes are typically not extended by a polymerase in the method, hybridizing sequences are designed based on the species-relevant sequence within the amplification product, which determines its Tm, %GC, and performance data. experimental obtained. In one embodiment, the hybridizing sequence of the first and second oligonucleotide probes is 9 to 20 nucleotide bases long. In an embodiment where the first and second hybridizing sequences in the target nucleic acid are separated by 0 bases, the sequence of the hybridizing regions of one of the oligonucleotide probes may correspond to one of the oligonucleotide primers and the hybridizing region. of the other oligonucleotide probe could correspond to the reverse complement of the other oligonucleotide primer. However, the length of the hybridizing sequences can be truncated to optimize the properties of the oligonucleotide probes for the desired modality of the method and to avoid any inhibitory effects in the event that all or part of step b) is performed simultaneously with step a). ). In the event that the first or second oligonucleotide probe encompasses a recognition sequence and cleavage site for any of the first or second restriction enzyme and said oligonucleotide probe is brought into contact with the sample simultaneously with the performance of step a) , the cleavage site within said probe is typically blocked, for example by inclusion of a modified internucleotide bond, eg a phosphorothioate bond, during chemical synthesis of the probe or introduction of a mismatch to eliminate said recognition sequence. There is considerable versatility other than the hybridizing regions to the sequence of the oligonucleotide probes and to any modified nucleotide bases, nucleotide linkages, or other modifications that they may comprise. Modified bases that can be chemically inserted into oligonucleotides to alter their properties and can be used in method modalities, such as 2-Amino-dA, 5-Methyl-dC, Super T®, QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ 2-Fluoro and Clamp G provide an increase in Tm, while others, such as Iso-dC and IsoG, can increase binding specificity without increasing Tm. Other modifications such as inosine or abasic sites can decrease the specificity of the binding. Modifications known to confer nuclease resistance include inverted dT and ddT and 03 spacers. Modifications may increase or decrease Tm and provide potential for event control of hybridization in embodiments of the method. The use of modified bases within the hybridizing regions of oligonucleotide probes provides an opportunity to improve the performance of oligonucleotide probes such as by increasing binding affinity without increasing the length of the hybridizing region. In one embodiment the modified bases within one or both of the oligonucleotide probes allows them to hybridize more effectively to, and thus outperform, any species within the amplification product with complementarity to the relevant single-stranded detection sequence. In embodiments where one of the first and second oligonucleotide probes has extension blocked at the 3' end and is not capable of being cleaved and contacted with the sample simultaneously with step a), typically said one probe The oligonucleotide will comprise an additional 5' region, which provides the opportunity for stabilization of the pre-sensor species as described (see Figure 2). In one such embodiment an oligonucleotide probe comprises the exact sequence of one of the oligonucleotide primers, but contains a modification at the 3' end to block its extension by strand displacement DNA polymerase and a single phosphorothioate internucleotide linker to block the restriction enzyme cleavage site. Such an embodiment simplifies assay design and ensures that no additional sequence motifs are introduced that could lead to non-specific background amplification. The first and second oligonucleotide probes producing the detective species are preferably provided at a level where the number of copies of the produced detective species is sufficiently above the detection limit of the media employed that said detective species is readily detected. Furthermore, the hybridization efficiency by the first and / or second oligonucleotide probe(s) is influenced by their concentration. Typically the concentration of an oligonucleotide probe contacted with the sample simultaneously with the performance of step a) can be similar to the concentration of the oligonucleotide primers, for example 10 to 200 nM, although this should be considered non-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, while in another embodiment the concentration of one or both oligonucleotide probes is provided at a lower concentration than one or both of the oligonucleotide probes. both oligonucleotide primers. In the case where one or both oligonucleotide probes contact QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ with the sample subsequent to performing the amplification step a), a higher concentration may be allowed as needed to effect the most efficient hybridization, without any consideration of inhibition to amplification step a). which can result Hybridization sequences are a key feature of both oligonucleotide primers and oligonucleotide probes for method performance. Hybridization refers to sequence-specific hybridization which is the ability of an oligonucleotide primer or probe to bind to a target nucleic acid or species within the amplification product by virtue of base pairing by hydrogen bonding between the complementary bases in the sequence of each nucleic acid. Typical base pairings are Adenine-Thymine (A-T), or AdenineUracil in the case of RNA or RNA / DNA hybrid duplexes, and Cytosine-Guanine (C-G), although a range of natural and unnatural base analogues are also known. of nucleic acids with particular binding preferences. Furthermore, in the present invention, the complementarity region of an oligonucleotide probe or primer need not necessarily fully comprise natural nucleic acid bases in a sequence with complete and exact complementarity to their hybridizing sequence in the target nucleic acid or species within. of the amplification product; rather for performance of the method the oligonucleotide probes / primers need only be capable of sequence-specific hybridization to their target hybridization sequence sufficiently to form the double-stranded sequence necessary for the correct performance of the method, including cleavage by restriction enzymes. and extension by strand displacement DNA polymerase. Therefore such hybridization may be possible without exact complementarity, and with unnatural bases or abasic sites. In one embodiment, the hybridizing regions of an oligonucleotide primer or oligonucleotide probe used in the method may consist of complete complementarity to the sequence of the relevant region of the target nucleic acid or species within the amplification product, or its reverse complementary sequence. , as appropriate. In other embodiments, one or more non-complementary base pairs exist. In some circumstances it may be advantageous to use a mixture of oligonucleotide primers and / or probes in the method. Thus, by way of example, in the case of a target nucleic acid comprising a single nucleotide polymorphism (SNP) having two polymorphic positions, a 1:1 mixture of oligonucleotide primers and oligonucleotide probes that differ may be employed. at that position (each component has complementarity to the respective base of the SNP). During the manufacture of oligonucleotides it is routine practice to randomize one or more bases during the synthesis procedure. One skilled in the art will understand that amplification procedures involving polymerases may suffer from non-specific background amplification such as that resulting from ab initio synthesis and / or primer-primer ligation. Although the method of the invention typically exhibits faster amplification when the amplicon length is designed to be as QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ shortens as much as possible, for example by minimizing primer annealing sequences, the gap between the first and second annealing sequences in the target nucleic acid, and the length of any stabilization regions, to the degree possible while still retaining function at the given reaction temperature. With shorter amplicons the non-specific background can be exacerbated due to the fact that all the sequence necessary to produce the amplification product species is provided by the oligonucleotide primers. In the event that an amplicon is produced in a non-target specific manner comprising both the first oligonucleotide primer and the second oligonucleotide primer linked via an ab initiOQ synthesized DNA primer junction, a false positive result in the method could occur. The use of two oligonucleotide probes in the present method allows a variety of method embodiments to encompass additional features to minimize any possibility of non-target specific background signal. Such modalities made it possible, through the use of two oligonucleotide probes, to present a substantial advantage over known methods in this regard. One approach is to separate the first and second hybridizing sequences in the target nucleic acid to provide a target-based sequence specificity check using the method's oligonucleotide probes. Thus in one embodiment, the first and second hybridizing sequences in the target nucleic acid are separated by 3 to 15 or 3 to 6 bases, for example 5, 7 or 11 bases. This gap between the primers presents the optimum size gap to provide additional species-specificity verification within the amplification product while still maintaining the improved speed of a short amplicon. Thus in one embodiment, in step b) any of the first or second single-stranded detection sequence in the at least one species within the amplification product includes at least 3 bases of the sequence corresponding to said 3 to 15 or 3 to 6 bases. For example, we have demonstrated the potential to distinguish a specific target-dependent amplification product from target-nonspecific background amplification products, as shown in Example 4 (Figure 8). In an alternative approach the concentration of the first and / or second oligonucleotide primers is decreased to reduce the likelihood of background resulting from ab initio amplification and primer-primer ligation. To ensure that the rate of amplification is maintained, additional oligonucleotide primers can be used that are blocked from extension at the 3' end by strand displacement DNA polymerase. In this embodiment, while the first and second unblocked oligonucleotide primers are available at sufficient concentration for the initial annealing and extension events to produce the target nucleic acid amplicon, subsequent amplification proceeds with the blocked primers, which are preferably provided at higher concentration, where cleavage of the blocked primers occurs before their extension and strand displacement to remove the blocking modification QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ of 3' and allows the amplification procedure to proceed without prejudice (see Figure 4). Thus in one embodiment, the sample is further contacted in step a) with: (a) a third oligonucleotide primer, said third primer comprising in the 5' to 3' direction a strand of the recognition sequence and site of cleavage for the first restriction enzyme and a region that is capable of hybridizing to the first hybridizing sequence in the target nucleic acid and wherein said third primer is blocked at the 3' end of the stretch by DNA polymerase; and / or (B) a fourth oligonucleotide primer, said fourth primer comprising in the 5' to 3' direction a strand of the recognition sequence and cleavage site for the second restriction enzyme and a region that is capable of hybridizing to the reverse complement of the second hybridization sequence into the target sequence and wherein said fourth primer is blocked at the 3' end of the extension by DNA polymerase. In a further embodiment, when the third oligonucleotide primer is present, excess of the first oligonucleotide primer is provided and when the fourth oligonucleotide primer is present, excess of the second oligonucleotide primer is provided. By reducing the concentration of the first and second oligonucleotide primers substantially, displaced by the presence of the third and fourth oligonucleotide primers, the maximum potential benefit in terms of eliminating target-independent background amplification is obtained. Other than the presence of the 3' modification to block polymerase extension which can be easily achieved through, for example, the use of a 3' phosphate or C-3 modification during oligonucleotide primer synthesis, The same design parameters as used for the first and second primers apply to the third and fourth primers. Modalities of the method of the invention that provide an increase in specificity and elimination of background amplification as described above, provide better stringency of sequence verification, enabling reactions to be performed at low temperatures without loss of specificity and / or enables increased multiplexing, where multiple reactions are performed for the simultaneous detection of multiple targets. The benefits of this stringent specificity also mean that the method can tolerate a wide temperature range and sub-optimal conditions (eg reagent concentrations) without loss of specificity. For example, we have performed the method with a 20% increase or decrease in the concentration of all components and we have performed the method with a substantial period at room temperature after performing the amplification in step a) in each case without observe no loss of specificity. Therefore such embodiments represent important advantages of the method of the invention over known methods and mean that it is ideally suitable for exploitation in a low cost and / or single use diagnostic device. Detection of the detected species in step c) can be accomplished by any technique that differentially detects the presence of the detected species from the other reagents and components. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ present in the sample. Alternatively the presence or level of the species to be detected can be inferred from the depletion of one or more reaction components such as the first or second oligonucleotide probe. Of a wide range of physicochemical techniques available for use in the detection of the detected species, priority is given to those capable of generating a sensitive signal that only exists after hybridization of the first oligonucleotide probe and the second oligonucleotide probe for the relevant species in the amplification product for use in the method. It will be apparent to a skilled person that there is a range of fluorometric or colorimetric dyes that can readily bind to the first oligonucleotide probe and form the basis of its detection, either visually or using instrumentation, such as absorbance or fluorescence spectroscopy. Thus in one embodiment, the portion that allows detection of the first oligonucleotide probe is a fluorometric or colorimetric dye or a portion that is capable of binding to a fluorometric or colorimetric dye such as biotin. Embodiments of the method employing colorimetric dyes have the advantage of not requiring an instrument to perform fluorescence excitation and detection and potentially of allowing the presence of the target nucleic acid to be determined visually. Colorimetric detection can be achieved by directly attaching a colorimetric dye or portion capable of binding a colorimetric dye to the first oligonucleotide probe prior to its use in the method, or alternatively by specifically attaching or linking the dye or portion to the probe fragment. after the split. For example, the first oligonucleotide probe may contain a biotin moiety that allows it to bind to a streptavidin-conjugated colorimetric dye for subsequent detection. One such example of a colorimetric dye that can be used in detection is gold nanoparticles. Similar methods can be employed with a variety of other intrinsically colorimetric moieties, of which a very large number are known, such as carbon nanoparticles, silver nanoparticles, iron oxide nanoparticles, polystyrene beads, quantum dots etc. A high extinction coefficient dye also provides potential for sensitive real-time quantification in the method. A number of considerations are taken into account when choosing the appropriate tint for a given application. For example, in modalities where visible colorimetric detection is intended to be performed in solution, it might generally be advantageous to choose particles of larger size and / or those with a higher extinction coefficient for ease of detection, while modalities incorporating a membrane For lateral flow detection, they could benefit from the ability of smaller particles to diffuse faster through a membrane. Although various sizes and shapes of gold nanoparticles are available, a number of other colorimetric moieties of interest are also available including polystyrene or latex based microspheres / nanoparticles. Particles of this nature also QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ are available in a number of colors, which may be useful for labeling and differentially detecting different species detected during method performance, or multiplexing the colorimetric signal produced in a detection reaction. Fluorometric detection can be accomplished through the use of any dye that, under the appropriate excitation stimulus, emits a fluorescent signal leading to subsequent detection of the species to be detected. For example, dyes for direct fluorescence detection include, without limitation: quantum dots, ALEXA dyes, fluorescein, ATTO dyes, rhodamine, and Texas red. In embodiments of the method that employ a portion of fluorescent dye attached to an oligonucleotide probe, it is also possible to perform detection based on fluorescence resonance energy transfer (FRET), as used in Taqman or qualitative PCR. strategies based on Molecular Beacons for the detection of nucleic acid, with which the signal could increase or decrease after the binding of the dye to the detected species. Generally, when using a fluorometric approach, a number of different detector devices can be used to record the generation of the fluorescent signal, such as for example CCD cameras, fluorescence scanners, fluorescence-based microplate readers or fluorescence microscopes. In a further embodiment the detection-enabling portion of the first oligonucleotide probe is an enzyme that produces a detectable signal, such as a colorimetric or fluorometric signal, upon contact with a substrate. It will be apparent to a skilled person that a number of enzyme substrate systems are available and are in everyday use in the diagnostic field, such as in ELISA and Immunohistochemical detection. Horseradish peroxidase (HRP) is an example. The use of an enzyme linked to the first oligonucleotide probe for detection of the species detected in step c), offers a number of potential advantages, such as increased detection sensitivity and increased signal control. developed through a separate step involving the addition of the substrate. Other suitable colorimetric enzymes could include: glucosyl hydrolases, peptidases or amylases, esterases (eg carboxyesterase), glucosidases (eg galactosidase), and phosphatases (eg alkaline phosphatase). This list should not be considered limiting in any way. In another approach, the presence of the detecting species in step c) is detected electrically, such as by a change in impedance or a change in the conductometric, amperometric, voltammetric or potentiometric signal, in the presence of the detecting species. Thus in one embodiment the detecting species is detected by a change in the electrical signal. The change in electrical signal can be facilitated by the portion that allows detection of the first oligonucleotide probe, such as a chemical group that leads to an increase in change in electrical signal. Since detection of the electrical signal may be so sensitive said detection portion may simply be an oligonucleotide sequence, although in certain embodiments the signal is increased. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ by the presence of chemical groups known to increase electrical signals, such as metals for example gold and carbon. While in one embodiment the change in electrical signal resulting from accumulation of the detector species can be detected in an aqueous reaction during amplification, in other embodiments the detection of the electrical signal is facilitated by localizing the detector species to a particular site for its detection, such as the surface of an electrochemical probe, wherein said localization is mediated by the second oligonucleotide probe. Other techniques that are routinely employed for the detection of nucleic acids such as the detector species and may also be employed for detection in the method include: mass spectrometry (such as MALDI or LC-TOF), spectroscopy or luminescence spectrometry, spectrometry or fluorescence spectrometry, liquid chromatography, and fluorescence polarization. In one embodiment, step c) produces a colorimetric or electrochemical signal using carbon or gold, preferably carbon. In one embodiment the detector species is detected by lateral flow of nucleic acid. Lateral nucleic acid flow, where nucleic acids are separated from other reaction components by diffusion through a membrane, typically made of nitrocellulose, is a rapid and low-cost detection method capable of being coupled with a range of signal readings, including colorimetric, fluorometric and electrical signals. Lateral flow of nucleic acid is very convenient for use in detecting the detector species in the method and offers a number of advantages. In one embodiment, nucleic acid lateral flow detection is performed wherein the first oligonucleotide probe within the detector species is used to bind a fluorometric or colorimetric dye and the second oligonucleotide probe within the detector species is used to locate said detector species. dye at a defined location on the lateral flow strip. In this way, rapid detection can be carried out with results visualized with the naked eye or by a reading instrument. Lateral flow nucleic acid can employ an antigen as the detector portion in the second oligonucleotide probe with the associated antibody immobilized on the lateral flow strip. As an alternative to the present method, sequence-specific detection via hybridization of the pre-detector species or the detector species on the lateral flow strip can be easily performed providing a simple, low-cost alternative to antibody-based assays with an improvement of the multiplexing potential. Known methods, such as SDA, that do not use the two oligonucleotide probes of the present method, typically generate double-stranded DNA products that are not available for detection based on sequence-specific hybridization. In contrast in the present method, the detector species is particularly prone to multiplexed detection, by virtue of the use of detection-based location-specific hybridization. can be QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ readily employ carbon or gold nanoparticles in nucleic acid lateral flow. The location of the detector species causes the local concentration of carbon or gold, causing the appearance of a black or red color, respectively. In one embodiment the first oligonudeotide probe contains a moiety, such as a biotin, that allows it to bind to a colorimetric dye prior to localization on the strip by sequence-specific hybridization. The spatial positioning of the detector species is closely associated with the technique employed for the detection of the detector species, since it allows, for example, hybridization based on the binding of the detector species at a particular location. In addition to facilitating rapid and specific detection, such physical binding may improve the use of the method in multiplexed detection of multiple different target nucleic acids. In one embodiment the second oligonudeotide probe is attached to a nucleic acid lateral flow strip or to the surface of an electrochemical probe, 96-well plate, beads, or array surface. Thus the at least one species within the amplification product becomes localized to the physical location of the second oligonudeotide probe that is easily detected after formation of the detector species at such location. Alternatively, it may be advantageous to use a single-stranded oligonudeotide as the portion attached to the second oligonudeotide probe that allows it to be attached to a solid material. In this way the sequence of the solid phase attached to the oligonudeotide can be defined independently of the target nucleic acid sequence to increase the efficiency of the binding. Thus, in one embodiment the portion that allows binding of the second oligonudeotide probe to a solid material is a single-stranded oligonudeotide. Said single-stranded oligonucleotide can be designed to have better affinity and hybridization efficiency to improve the performance of the method. For example, in certain embodiments of the method, rather than attaching the second oligonucleotide probe to the lateral flow strip directly, a separate oligonucleotide with an optimized sequence for hybridization on the strip is used that is capable of efficient hybridization to the oligonucleotide portion. single stranded strand present within the second oligonudeotide probe. In several investigations we have significantly improved the performance of the nucleic acid lateral flow method by using a single-stranded oligonudeotide as the binding portion of the second oligonudeotide probe, which provides hybridization on the strip of the sequence to be augmented. For example, a G-C rich sequence can be used for hybridization on the strip, or a longer sequence with higher Tm can be used, which complements the length of the second oligonudeotide probe. Alternatively, said single-stranded oligonucleotide portion may comprise one or more modified bases or internucleotide linkages to increase their affinity, such as a PNA, LNA, or G-clamp. We have observed that when a repeat sequence motif is employed in the single-stranded oligonucleotide portion, a striking increase in hybridization efficiency is observed which is not predicted by its predicted Tm. so in one QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ embodiment the sequence of the single-stranded oligonucleotide portion comprises three or more repeat copies of a 2 to 4 base DNA sequence motif. For example, in several investigations employing such a sequence motif we have observed a substantial increase in the sensitivity of nucleic acid lateral flow detection, often with a 100-fold or greater signal increase. Thus in one embodiment where the presence of the species to be detected is detected by nucleic acid lateral flow, nucleic acid lateral flow uses one or more nucleic acids that are capable of sequence-specific hybridization to the moiety that allows the binding of the nucleic acid. second oligonucleotide probe to a solid material. A further advantage is conferred by uncoupling of the target nucleic acid sequence from the solid material for binding or from the detection medium, this can be enabled by the use of the single-stranded oligonucleotide as the detection portion within the first oligonucleotide probe and / or or the binding portion with the second oligonucleotide probe. In this way the relevant solid material for binding, or device containing said solid material, such as the nucleic acid lateral flow strip, and / or detection means, can be optimized and defined without considering the nucleic acid sequence. target to be detected. Such universal detection apparatus can be used from application to application and from target to target without needing to be altered. For example, a nucleic acid lateral flow strip can be defined with imprinted lines corresponding to a compatible set of oligonucleotide sequences that have the ability to efficiently hybridize to the strip and without unintentional cross-talk, optimized and efficiently fabricated regardless of the development of the oligonucleotide primers and probes of the method for the detection of multiple target nucleic acid sequences. In a number of embodiments the detection can be performed in a quantitative manner. Thus, the level of the single-stranded target nucleic acid in the sample can be quantified in step c). Quantification can be accomplished, for example, by measuring the detected species colorimetrically, fluorometrically, or electrically, over the course of the reaction at multiple time points rather than a single end point. Alternative strategies for quantification include sequential dilution of the sample, analogous to droplet digital PCR. In a further embodiment the level of the single-stranded target nucleic acid in the sample can be determined semi-quantitatively. For example, where the intensity of a colorimetric signal on a nucleic acid lateral flow strip might correspond 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 target nucleic acid must exceed a certain defined copy number in order to overcome the inhibitor and produce a detectable copy number of the detectable species. In the method of the invention the second oligonucleotide probe is attached to a QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ solid material or to a portion that allows its attachment to a solid material. Optionally, in embodiments, one or more of the other oligonucleotide primers and probes may also be attached to a solid material or to a portion that allows binding to a solid material. It will be apparent to a skilled individual that attachment of oligonucleotides to a solid material can be accomplished in a variety of different ways. For example, a number of different solid materials are available that have or can be bound or functionalized with a sufficient density of functional groups to be useful for the purposes of binding or reacting with appropriately modified oligonucleotide probes. In addition, a wide range of profiles, sizes, and shapes of such solid materials are available, including beads, resins, surface-coated plates, slippers, and capillaries. Examples of such solid materials used for covalent attachment of oligonucleotides include, without limitation: glass sliders, glass beads, ferrite core polymer-coated magnetic microbeads, silica microparticles or magnetic silica microparticles, silica-based microcapillary tubes , 3D reactive polymer sliders, 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 additional advantage that a functionalized oligonucleotide can be covalently attached during the polymerization reaction between monomers (eg, acrylamide monomers) that are used to produce the polymer. A functionalized oligonucleotide is included in the polymerization reaction to produce a solid polymer containing the covalently linked oligonucleotide. Such polymerization represents a highly efficient means of binding oligonucleotide to a solid material with control over the size, profile, and shape of the produced solid material attached to the oligonucleotide. Typically to attach an oligonucleotide probe to any such solid material, the oligonucleotide is synthesized with a functional group at either the 3' or 5' end; although functional groups can also be added during the oligonucleotide production process at almost any position on the base. A specific reaction can then be performed between the functional group(s) within an oligonucleotide and a functional group on the relevant solid material to form a stable covalent bond, resulting in an oligonucleotide bound to a solid material. Typically such an oligonucleotide could be attached to the solid material at either the 5' or 3' end. By way of example, two commonly used and reliable ligation chemistries utilize a thiol (SH) or amine (NH3) group and functional group on the oligonucleotide. A thiol group can react with a maleimide moiety on the solid support to form a thioester bond, while an amine can react with a modified carboxylic acid succinimidyl ester (NHS ester) to form an amide bond. You can also use a number of other QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ chemicals. As well as the chemical conjugation of an oligonucleotide probe to a solid material, it is possible and potentially advantageous to directly synthesize oligonucleotide probes on a solid material for use in carrying out the method. In other embodiments the second oligonucleotide probe is attached to a portion that allows it to be attached to a solid material. One strategy is to employ an affinity binding method whereby a moiety that allows for specific binding can be attached to the oligonucleotide probe to facilitate its binding to the relevant affinity ligand. This can be done, for example, using antibody-antigen binding or an affinity tag, such as a poly-histidine tag, or using nucleic acid-based hybridization where the complementary nucleic acid is attached to a solid material, for example a nitrocellulose nucleic acid lateral flow strip. One such exemplary moiety is biotin, which is capable of high affinity binding to streptavidin or avidin which itself is bound to beads or other solid surfaces. 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 series of separate steps a), b), and c) are performed, using different oligonucleotide primers and oligonucleotide probes for each of the two or more target nucleic acids, said separate steps may be performed simultaneously. . For example, in one embodiment, one set of oligonucleotide primers and oligonucleotide probes could be used for the detection of one target nucleic acid in a sample and another set of oligonucleotide primers and oligonucleotide probes could be used for the detection of another. target nucleic acid in the same sample. Each detection of the detecting species produced from the two or more different sets of primers / probes could be coupled to a particular signal, such as different dyes or colorimetric or fluorometric enzymes, to allow multiplexed detection. Alternatively, multiplexed detection can be achieved by binding the second oligonucleotide probe to a solid material, directly or indirectly through a portion that allows it to bind to a solid material. Such an approach uses the physical separation of the detecting species produced by the different series of steps a), b) and c), rather than relying on a different means of detection. Thus, for example, a single nucleic acid lateral flow dye could be used to detect multiple different target nucleic acids where each different detect species produced locates to a particular printed line on the lateral flow strip and hybridization based on from the direct or indirect sequence to the second oligonucleotide probe forms the basis of differential detection. Alternatively, an electrical detection array can be used where multiple different second oligonucleotide probes bind to a particular region of the array and so in a multiplexed reaction where multiple different detect species are produced at the same time, each detect species becomes localized. via hybridization to a discrete region of the array allowing multiplexed detection. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ Previous detection procedures, such as nucleic acid lateral flow and electrical detection, and their ability to easily detect multiple different target nucleic acids within the same sample, are enabled by the present method's intrinsic requirement for the two oligonucleotide probes. As such they could powerfully demonstrate the advantages of the method of the invention over known methods. The current invention is of wide utility in various fields and applications that require the detection of a target nucleic acid of defined sequence in a sample. It represents a fast, inexpensive and convenient means of determining the presence of a target nucleic acid sequence within a sample. By means of a list of applications that is not in any way limiting, we contemplate that the invention could be of value in fields such as; diagnostic, forensic, agricultural, animal health, environmental, defense, human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics or pharmacokinetics and microbiology, clinical, and biomedical research. Suitably the sample is a biological sample such as a human sample. The sample can be a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample. Target nucleic acid detection can be used for the diagnosis, prognosis, or monitoring of disease or a disease state such as 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 cancer, including but not limited to colorectal cancer, lung, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, kidney cancer, stomach cancer, or melanoma, or in the fields of human genetic testing, prenatal tests, examination of blood contamination, pharmacogenetics or pharmacokinetics. The invention is capable of use with a wide array of sample types, such as, for example: nasal swabs, nasopharyngeal swabs, throat swabs, cheek swabs, blood or a derived sample. blood, urine or a sample derived from urine, sputum or a sample derived from sputum, stool or a sample derived from stool, cerebrospinal fluid (CSF) or a sample derived from CSF, and gastric fluids or a sample derived from gastric fluids, human or animal samples derived from any form of biopsy of tissue or bodily fluid. We have also performed the method on a wide range of samples containing at least 10-20% of the following clinical specimens: VTM nasal swab, VTM nasopharyngeal swab, thin prep medium, liquid Amies throat swab, HSV ulcer in M4 medium, synovial fluid, sputum processed via NaOH QHRnnn / ιζηζ / Β / γίΛΐ 2M / isopropanol followed by DNA capture beads, TE rectal swab, stool sample processed by homogenization and DNA capture beads, CSF, APTIMA swab, amniotic fluid, oral swab in Amies liquid, urine, VRE swab in TE, pleural fluid, whole blood, K2EDTA plasma, L.Heparin plasma, and blood serum. These experiments have demonstrated the remarkable versatility of the method for different 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 specimens, such as PCR-inhibiting heparin and phytic acid, and thus demonstrates the potential for using the method in a low-cost or single-use device. use without any requirement for complex sample preparation procedures. The target nucleic acid can be (a) viral or derived from viral nucleic acid material (b) bacterial or derived from bacterial nucleic acid material (c) circulating cell-free DNA released from cancer cells (d), DNA cell free, circulating, released from fetal cells or (e) microRNA or microRNA derivative inter alia. The single-stranded target nucleic acid for the method may be natural or non-natural. The target nucleic acid can be generated in situ produced from natural nucleic acid prior to carrying out the method. A single-stranded target nucleic acid for the method may be prepared by one or more additional steps performed before or simultaneously with step a), said additional steps may encompass one or more enzymes such as polymerases and restriction enzymes. The generation of target nucleic acid for the method thus has a number of potential advantages, such as allowing even more highly multiplexed and / or background-breaking ab initio assays. A highly specific conversion of nucleic acid material in a biological sample can, for example, be performed without amplification prior to amplification in step a). The sample can be, for example, treated, purified, buffer-exchanged, exorn-captured, partially depleted of contaminating material, and / or converted to a single-stranded target nucleic acid for the method containing one or more modified dNTPs. Provided that the actual target nucleic acid in the sample to be detected is converted to the surrogate target nucleic acid for method performance with reliable conversion (which can be <1:1, 1:1 or 1:>1 ie possibly with some element of amplification) then detection of the surrogate target nucleic acid will allow the actual nucleic acid to be detected and / or quantified. Furthermore, the production of a surrogate target from a natural target can be used in this manner to generate in a method-specific manner a target nucleic acid with any desired sequence. In one embodiment where the single-stranded target nucleic acid is derived from double-stranded DNA after dissociation of the two strands, for example by strand invasion, two complementary single-stranded nucleic acid targets are present and can be amplified and detected in a reciprocal method by the same oligonucleotide primers and probes. In QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ where the target is the genome of a single-stranded RNA virus -ve strand, the transcript of the -ι-ve strand may also be present in the sample and either or both strands may be amplified and detected as the single-stranded target nucleic acid in the method using the same primers and oligonucleotide probes. It is also contemplated that the present invention has the potential to be useful in screening samples for cell-free DNA and epigenetic modifications such as, for example, CpG methylation of DNA sequences. Such epigenetic modification of target genes associated with a particular cancer can serve as useful biomarkers in a number of diseases and disease states. Given the growing appreciation of the importance of epigenetic modification in human disease, there is potential for the present invention to be used to specifically assess epigenetic modification of particular target nucleic acid biomarkers based on differential DNA polymerase activity. strand displacement and / or restriction enzymes. Thus, in one embodiment, the target nucleic acid contains a site of epigenetic modification, such as methylation. Alternatively, the actual nucleic acid used to produce a surrogate target nucleic acid for carrying out the method, as described above, contains a site of epigenetic modification. A further aspect of the invention relates to kits for use in the detection of nucleic acids of defined sequence in a sample. Thus the invention also provides a kit comprising the following: a) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridizing to a first hybridization sequence to a single-stranded target nucleic acid of defined sequence, and said second primer comprises in the 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region that is 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 that is not a nicking enzyme and is capable of recognizing the recognition sequence of and cleaving the first primer cleavage site and a second restriction enzyme that is not a nicking enzyme and is capable of recognizing the sequence recognizing and cleaving the second primer cleavage site; c) a strand displacement DNA polymerase; d) dNTPs; e) one or more modified dNTPs; QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ f) a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species in the amplification product produced in the presence of said target nucleic acid and that is linked to a portion that allows its detection ; and g) a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in said at least one species in the amplification product and that is attached to a solid material or to a portion that allows attachment to a solid material. In one embodiment one of the first and second oligonucleotide probes in the kit is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, for example due to the presence of one or more sequence mismatches and / or one or more modifications such as a phosphorothioate bond. In one embodiment one of the first and second oligonucleotide probes of the kit has 5 or more bases of complementarity to the region of hybridization or the reverse complement of the region of hybridization of the first or second primer. In another embodiment the first oligonucleotide probe of the kit has some complementarity, for example 5 or more bases of complementarity, to the region of hybridization of one of the first and second oligonucleotide primers, and / or the second oligonucleotide probe of the kit has part of complementarity, eg 5 or more bases of complementarity, to the reverse complement of the hybridization region of the other of the first and second oligonucleotide primers. In other embodiments the first and / or second oligonucleotide probes may have some complementarity or inverse gap complementarity between the first and second hybridizing sequences on the target nucleic acid as described above. The kit can also comprise a reverse transcriptase. The kit may further comprise means for detecting the presence of a detecting species produced in the presence of the 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 a surface array, and / or a fluorometric or colorimetric stain and / or a device for the detection of a change in electrical signal, and / or carbon or gold. In various embodiments, the target nucleic acid and the kit components, 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 QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ oligonucleotide and / or any of the first or second single-stranded detection sequence in the at least one species within the amplification product comprised in the kit are as defined herein for the methods of the invention. For example, the kit may comprise any combination of the characteristics of such components described herein, such as, without limitation, the following: One of the first and second oligonucleotide probes is blocked from extension at the 3' end by polymerase of DNA and is not capable of being cleaved by either the first or second restriction enzymes optionally due to the presence of one or more sequence mismatches and / or one or more modifications such as a phosphorothioate bond; the first restriction enzyme and the second restriction enzyme are the same restriction enzyme; the one or more modified dNTPs is an alpha thiol modified dNTP; the portion that allows detection of the first oligonucleotide probe is a fluorometric or colorimetric dye or a portion that is capable of binding to a fluorometric or colorimetric dye, such as biotin; the portion that allows binding of the second oligonucleotide probe to a solid material is a single-stranded oligonucleotide, optionally comprising three or more repeat copies of a 2 to 4 base ADB sequence motif; the first and second oligonucleotide primers comprise a stabilizing sequence upstream of the restriction enzyme recognition sequence and cleavage site, such as at the 5' end, and for example 5 or 6 bases in length; the hybridizing region of the first and / or second oligonucleotide primers is between 6 and 30, eg 9 and 16, bases in length; and, the first and second hybridizing sequences in the target nucleic acid are separated by 0 to 15 or 0 to 6 bases, in certain embodiments they are separated by 3 to 15 or 3 to 6 bases, for example 5, 7 or 11 bases , or overlap such as by 1 to 2 bases. The kits may comprise means for detecting the presence of a detectable species produced in the presence of the target nucleic acid, such as a nucleic acid lateral flow strip. In a further embodiment, the kit further comprises the third and / or fourth oligonucleotide primers as defined herein. Kits may also include reagents such as reaction regulators, salts eg divalent metal ions, additives and excipients. Kits according to the invention may be provided together with instructions for carrying out the methods according to the invention. The invention also provides for the use of the kits of the invention for the detection of a single-stranded target nucleic acid of defined sequence in a sample. It is to be understood that all optional and / or preferred embodiments of the invention described herein in relation to the methods of the invention also apply in relation to the kits of the invention and the use thereof, and vice versa. As previously mentioned the methods and kits of the invention are ideally QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ suitable for use in a device, such as a single-use diagnostic device. Thus the invention also provides a device containing a kit as described above, in particular a kit comprising means for detecting the presence of a detector species produced in the presence of the target nucleic acid, such as a nucleic acid lateral flow strip. . The device may be a powered device, for example an electrically powered device, the device may also comprise heating means and may be a self-contained device, ie a device that does not require an auxiliary test instrument. The method of the invention can also be used independently of detection step c) to amplify a nucleic acid signal from a target nucleic acid of defined sequence, such a method can be used, for example, if the amplified signal is to be stored and / or transport for detection of the target nucleic acid at a future date and / or alternative location if required. The amplified signal comprises the pre-detector species or detector species produced through carrying out the method. Thus, in a further embodiment, the invention provides a method of amplifying a nucleic acid signal of a target nucleic acid of defined sequence in a sample comprising steps a) and all or part, for example part i. or ii., from step b) of the method of the invention. The invention also provides for the use of the kits of the invention to amplify a nucleic acid signal from a target nucleic acid of defined sequence as defined above. It is to be understood that all the optional and / or preferred embodiments of the invention described herein in relation to the methods of the invention for the detection of the presence of a target nucleic acid of defined sequence in a sample also apply in relation to the method for the amplification of a nucleic acid signal from a target nucleic acid of defined sequence. The following examples serve to further illustrate various aspects and embodiments of the methods described herein. These examples are not to be considered limiting in any way. EXAMPLES materials v methods The following materials and methods are used in the subsequent examples unless otherwise indicated. Oligonucleotides: Except as otherwise indicated, custom oligonucleotides were fabricated using the phosphoramidite method by Integrated DNA Technologies. Lateral flow of nucleic acid. Carbon nanoparticles were conjugated via non-covalent adsorption to various biotin-binding proteins, eg streptavidin. Typically, it QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ prepared a suspension of colloidal carbon in Borate buffer followed by sonication using a probe sonicator. The carbon was subsequently adsorbed to the biotin-binding protein by incubation at room temperature. The carbon was used either directly in the reaction mixtures or applied to fiberglass conjugate pads. Lateral flow strips were constructed by combining a conjugate pad containing lyophilized sugars and additives used to improve visual appearance with a sample pad, nitrocellulose membrane, and adsorbent pad (Merck Millipore) following the manufacturer's guidelines. Prior to use in the lateral flow strips, the relevant oligonucleotide(s) containing the reverse complement of the sequence to be detected in the method were printed onto the nitrocellulose membrane at a defined location and attached to the membrane via UV cross-linking. EXAMPLE 1 Carrying out the method where the second oligonucleotide probe is attached to a solid material, a nitrocellulose flow-through strip This example demonstrates performing the method where the second oligonucleotide probe is attached to a solid material, a nitrocellulose lateral flow strip, and the first oligonucleotide probe is not contacted with the sample simultaneously with performing the sample step. amplification a). The first oligonucleotide primer with a total length of 24 bases was designed to comprise in the 5' to 3' direction: A 7 base stabilizing region; the 5 bases of the recognition sequence for a restriction enzyme that is not a nicking enzyme; and a 12 base hybridizing region comprising the reverse complementary sequence of the first hybridizing sequence in the target nucleic acid. The second oligonucleotide primer was designed to contain the same stabilization region and restriction enzyme recognition sequence, but with the 12 base hybridizing region capable of hybridizing to the reverse complement of the second hybridizing sequence in the target nucleic acid. In this example the first restriction enzyme and the second restriction enzyme are the same restriction enzyme. The restriction enzyme is an asymmetric double-stranded cleavage restriction enzyme with an upstream cleavage site downstream of its 5 base recognition sequence. The first and second hybridization sequences in the target nucleic acid are separated by 1 basis. The oligonucleotide primers were designed using the target nucleic acid so that the nucleotide base downstream of the cleavage site in the reverse complement of the primers is Adenosine so that the alpha thiol dATP is used as the modified dNTP in QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ the method. A phosphorothioate modification is inserted by strand displacement polymerase to block cleavage of said reverse complementary strand. The first oligonucleotide probe with a total length of 20 bases was designed to comprise in the 5' to 3' direction: A 12 base region of complementarity to at least one species in the amplification product; a 6 base neutral spade region; and a 3' biotin modification added during synthesis wherein said biotin modification allows binding of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles. Carbon adsorbed to a biotin-binding protein was prepared and saturated with the first oligonucleotide probe. The second oligonucleotide probe with a total length of 49 bases was designed to comprise, in the 5' to 3' direction: A neutral spacer comprising 10 X Thymidine bases; 3 X repeats of a 13 base region capable of hybridizing to the second single-stranded detection sequence downstream of the first single-stranded detection sequence in said at least one species in the amplification product. Approximately 30pmoles of said second oligonucleotide probe were printed on the nucleic acid flow strip. The reactions were prepared containing; 1.6pmoles of the first primer; O.pmoles of the second primer; 250μΜ Sp-isomer of 2'-Deoxyadenosine-5'-O-(l-thiotriphosphate) (Sp-dATP-a-S) from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; 2U of restriction enzyme; and 2U of a Bacillus strand displacement DNA polymerase. The target nucleic acid (a target single-stranded DNA) was added at various levels (++ = 1 mol, + = 10 zmol, NTC = no target control) in a total reaction volume of 10 µΙ in an appropriate reaction buffer. The reactions were incubated at 45°C for 7 or 10 min. 6.5μΙ of the finished reaction mix was then added to 60μΙ of the sidestream buffer containing 0.056 mgml'1 of the conjugated carbon before being loaded onto the nucleic acid lateral flow strip with the second oligonucleotide probe attached to it. in a printed line. Figure 5 presents a photograph of the lateral flow strips obtained in the exemplary embodiment. An arrow indicates the position where the second oligonucleotide probe has been printed on the nitrocellulose strip and then where the positive signal appears. A light black line corresponding to the presence of the carbon signal alone was observed in the presence of the target nucleic acid at both target levels and at both time points demonstrating the rapid and sensitive detection of the target nucleic acid sequence by the method of the invention. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ EXAMPLE 2 Carrying out the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase v is not capable of being cleaved by either the first or second restriction enzyme v is contacted with the sample in step a) This example demonstrates the performance of embodiments of the methods where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and is placed in contact with the sample simultaneously with the performance of step a). In such modalities, we have not observed any significant inhibition of amplification rate, indicating that the pre-detector species accumulates in real time without disturbing the optimal cyclic amplification procedure. Not only did we not observe any inhibitory effect on the amplification procedure in said modalities, but we observed a surprising improvement in the signal produced corresponding to an increase in the amount of the species detected, of at least 100 times. EXAMPLE 2.1: A variant of the assay used in Example 1 was designed by exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by any of the first or second restriction enzyme and is placed in contact with the sample simultaneously with the performance of step a). The same oligonucleotide primers, restriction enzyme, dNTPs, modified dNTPs, and polymerase were used as used in Example 1, however, an alternative first oligonucleotide probe was designed with a total length of 21 bases spanning in the direction 5' to 3': a 5' biotin modification; an 8 base neutral region; a 13 base region capable of hybridizing to at least one species in the amplification product; and a 3' phosphate modification, where the biotin modification allows binding of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the phosphate modification blocks its extension by translocation DNA polymerase. chain. Carbon adsorbed to a biotin-binding protein was prepared and saturated with the first oligonucleotide probe. A second alternative oligonucleotide probe with a total length of 51 bases was designed comprising, in the 5' to 3' direction: A 14 base region capable of hybridizing to the second single-stranded detection sequence 5' of the first sequence detecting single-stranded in said at least one species in the amplification product; a 6 base neutral spade sequence; a 14 base hybridizing region repeat; a second 6 base neutral spade sequence; and 10 X of a Thymidine-based sword. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ Approximately 30pmoles of said second oligonucleotide probe were printed on the nucleic acid flow strip. The reactions were prepared with: 0.8pmoles of the first primer; 0.8pmole of the second primer; 0.6pmole of the first oligonucleotide probe; 300μΜ of Sp-dATP-α-S; 60μΜ each of dTTP, dCTP and dGTP; 2U of restriction enzyme; and 2U of a Bacillus strand displacement DNA polymerase. The target nucleic acid (a target single-stranded DNA) was added at various levels (++ = 1 mol, + = 10 zmol, NTC = no target control) in a total reaction volume of ΙΟμΙ in an appropriate reaction buffer. The reactions were incubated at 45°C for 6 min. 5μΙ of the completed reaction mix was then added to a 60μΙ sidestream buffer containing 0.03 mgml·1 conjugated carbon before being loaded onto the nucleic acid lateral flow strip. A control reaction was performed to demonstrate that the detect species is not produced when the first oligonucleotide probe was not present during the reaction. The equivalent level (0.opmoles) of the probe was added to said control after step a) to control for any undesired impact of the presence of the probe during detection of the lateral flow strip. Figure 6A presents a photograph of the nucleic acid lateral flow strips after development. The clear signal corresponding to the deposition of the carbon nanoparticles was observed at both target levels when the first oligonucleotide probe was provided during the reaction. As expected, a signal was not detected at any target level when the first oligonucleotide was not provided during the reaction. This experiment clearly demonstrates the potential to substantially increase the production of detectable species in embodiments of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by any of the first or second restriction enzyme and placed in contact with the sample simultaneously with the performance of step a). It is notable that, in contrast to Example 1, an equal concentration of the first and second oligonucleotide primers was provided, enabling faster amplification. EXAMPLE 2.2: A separate assay was designed below to demonstrate the versatility of such method modalities with a completely different target nucleic acid. Oligonucleotide primers and oligonucleotide probes were designed for the relevant target nucleic acid, single-stranded DNA, in a similar manner as described in Examples 1 and 2.1. The reactions were prepared with; 0.8pmole of the first primer; 0.4pmole of the second primer; 0.6pmole of the first oligonucleotide probe; 300μΜ of Sp-dATP-α-S; 60μΜ each of dTTP, dCTP and dGTP; 2U of restriction enzyme; and 2U of a Bacillus strand displacement DNA polymerase. The target nucleic acid (a target single-stranded DNA) was QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ added at various levels (+=1 mol, NTC=no target control) in a total reaction volume of ΙΟμΙ in an appropriate reaction buffer. The reactions were incubated at 45°C for 6 min. 5μΙ of the completed reaction mix was then added to a 60μΙ sidestream buffer containing 0.08 mgml·1 conjugated carbon before being loaded onto the nucleic acid lateral flow strip. A control reaction comprising a truncated variant of the first oligonucleotide probe was performed which was also contacted with the sample simultaneously with the performance of step a). Figure 6B presents a photograph of the nucleic acid lateral flow strips after development. The clear positive signal was visible in the target nucleic acid present and not in the no-target control demonstrating correct assay design and performance and the robust potential of method embodiments where the first oligonucleotide probe is blocked for extension in the target nucleic acid. 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and brought into contact with the sample simultaneously with the performance of step a). As expected only a very minimal signal was observed in the control assay employing a truncated form of the first oligonucleotide probe, demonstrating the requirement for correct hybridization of the first oligonucleotide probe simultaneously with performing amplification in step a) for the efficient production of the species detected. EXAMPLE 3 Implementation of the method where 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 for the detection of two or more different target nucleic acids of defined sequence in a sample. The use of two oligonucleotide probes in addition to the primers in the method provides a comprehensive approach to the detection of the amplification product in the method that is ideally suitable for the detection of two or more different target nucleic acids in the same sample. This example demonstrates the ability to differentially detect the alternate detectable species based on sequence-specific hybridization of the second oligonucleotide probe. Firstly, to demonstrate the capability of the method to be used for the detection of two or more different target nucleic acids we developed compatible sets of oligonucleotide primers and probes for the detection of two different targets (A and B). In each case the first oligonucleotide probe was designed to contain the following features in the 5'-3' direction: a 5' biotin modification, a 7 base stabilization region, all 5 bases of an endonuclease recognition site restriction, a region of 11 - 13 bases QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ complementary to the 3' end of target A or B comprising a phosphorothioate bond at the restriction enzyme cleavage site, and a 3' phosphate modification. The second oligonucleotide probe was designed to contain in the 5'-3' direction: A 12-14 base region complementary to the 5' end of target A or B, a 5 X Thymidine base neutral spacer, and a portion of 12 base single-stranded oligonucleotide as the portion that allows attachment of the second oligonucleotide probe to a solid material. The sequence of the single-stranded oligonucleotide binding portion for each target was designed using a different sequence to allow binding of each species to be detected at a different location on the lateral flow strip. Lateral flow nucleic acid strips were prepared containing discrete 30pmole spots of an oligonucleotide containing the reverse complementary sequence to each single-stranded oligonucleotide detection portion at separate locations. Reactions were assembled containing: 0.5pmoles of the first oligonucleotide probe for target A and target B; 0.5pmole of the second oligonucleotide primer for target A and B, in 65pl of an appropriate buffer containing 0.032mgml'1 carbon adsorbed to a biotin-binding protein. Different levels of each target (+ = 0.lpmol; ++ = lpmol) were added to separate reactions individually and both targets were added together. A No Target Control (NTC) was also performed. Figure 7A presents a photograph of the lateral flow strips obtained in the experiment. Light black dots corresponding to carbon deposition containing the detected species were observed at both target levels and for both assays. Furthermore, when both reactions were performed at the same time, the signal corresponding to both targets A and B was observed. No background signal or crosstalk was observed between the different assays. To demonstrate the robustness of the method, a further experiment was carried out to develop three separate assays to demonstrate the potential of the method for the detection of three different target nucleic acids of defined sequence in a sample. A similar methodology was employed as described above. Figure 7B presents a photograph of the obtained lateral flow strips. Pl, P2 and P3 targets 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 on the nucleic acid lateral flow strip in separate lines. The black signal indicates the deposition of the detected carbon-bound species located at the location expected in all cases for rapid sensitive detection without unintentional cross-talk between the assay or any background signals. An equivalent experiment comprising four separate assays demonstrates the potential of the method for the detection of four different target nucleic acids (P1, P2, P3 and P4) of defined sequence in a sample with the results shown in Figure 7C. In this four-target experiment, P4 was present on all QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ reactions as a positive control and the other targets were individually added to separate reactions. Photographs of the lateral flow strips showed light black bands developed at the expected locations, corresponding to the presence of the relevant carbon-bound detectara species. Such multiplexed assays demonstrate the potential of the method to be used for diagnostic tests for diseases that are caused by a number of different pathogens where detection of the presence of the control assay detected species indicates that the method has been successfully performed and visualization of one or more of the other detectable species on the lateral flow strip indicates the presence of the relevant causative pathogen(s) in an appropriate clinical specimen. While it would be rare in such diagnostic applications, such as in the field of infectious diseases, to observe co-infections where more than one pathogen is present in the same specimen, the method of the invention is highly versatile for any combination of targets. in a multiplexed reaction to be detected. Figure 7D presents the results of an experiment where different combinations of four targets (Pl, P2, P3 and P4) are added. The ability to detect each target individually and the detection of three other targets when each target is missed without non-specific background demonstrates the remarkable detection specificity of the method of the invention. In what was described above and in several other experiments, we have also performed multiplexed assays for the detection of 3-5 targets at very low target concentrations, eg 1zmol (600 copies) or 17ymoles (10 copies). In this example, we have clearly demonstrated the potential of the method to detect the presence of two or more different target nucleic acids of defined sequence in a sample, and its potential for rapid signal detection at low cost, for example by lateral flow of nucleic acid. It is an unusual and advantageous feature of the method of the invention that two or more different target nucleic acids of defined sequence can be easily detected in the same sample. For each additional target to be detected, an additional set of oligonucleotide primers is required, which in prior art methods without temperature cycling presents a significant challenge for detecting the presence of two or more different target nucleic acids, because the primers additions lead to an increased propensity to form non-specific amplification products. In the method of the invention, this challenge is overcome by increased specificity, such as that resulting from the use of modified bases, better enzyme selection, and the formation of a detected species using oligonucleotide probes that exploit additional instances of sequence specific hybridization. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ EXAMPLE 4 Carrying out the method where the first and second hybridization sequences in the target nucleic acid are separated by 5 bases This example demonstrates carrying out the method where the first and second hybridizing sequences in the target nucleic acid are separated by 5 bases. The ability to use sequence derived from the target that is not present in the oligonucleotide primers and only occurs 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, provide the potential for increased specificity in embodiments of the method that can overcome any background signals arising from ab initio synthesis and primer-primer junction. In such embodiments the sequence-specific hybridization of the first or second oligonucleotide probe is designed to exploit the gap between the two hybridization regions so that the detector species is produced only when the amplification product contains the correct target derived sequence. In this example we designed a range of assays to demonstrate hybridization of the second oligonucleotide probe to several different amplification products that differ only in the gap sequence between the first and second hybridization sequences within the target nucleic acid. The second oligonucleotide probe was designed to contain an 11 base hybridizing region for the at least one species in the amplification product at its 5' end. Said region was formed from a sequence of up to 7 bases that is the reverse complementary sequence of the first oligonucleotide primer and a sequence of 5 bases that is the reverse complementary sequence to the additional target derived sequence in the amplification product derived from the gap between the primers. two primers. The second oligonucleotide probe also contained in the 5' to 3' direction a 5 X thymidine base neutral spacer and a 12 base single-stranded oligonucleotide portion for binding to solid material. A nitrocellulose nucleic acid flow strip imprinted with 30pmoles of an oligonucleotide with the reverse complementarity sequence of said portion was prepared. The first oligonucleotide probe was designed to contain the same sequence as the second oligonucleotide primer but with a 5' biotin modification, a 3' phosphate modification, and a phosphorothioate internucleotide linkage at the position of the enzyme cleavage site. of restriction. Four different artificial target nucleic acid sequences (TI, T2, T3 and T4) were designed, each of which had the exact sequence corresponding to the first and second hybridization sequences, but which differed by five bases between the first and second. second hybridization sequences: TI contains the correct bases for detection with complete complementarity to QbRnnn / ιζηζ / Β / γίΛΐ the 11 base hybridization region of the second oligonucleotide probe; T2 contains four mismatches of the five void bases; T3 was designed so that four bases of the five bases in the gap are deleted and therefore the amplification product species is four bases shorter. T4 contains two mismatches of the five void bases. Reactions were assembled containing: 3.6pmoles of the oligonucleotide first primer; 1.8pmole of the second oligonucleotide primer; 2.4pmoles of the first oligonucleotide probe; 300μΜ of Sp-dATP-a-S, 60μΜ of dTTP, dCTP, dGTP; 12U of restriction enzyme; 12U of a Bacillus strand-displacing DNA polymerase in a total reaction volume of 60μΙ in an appropriate reaction buffer, lamol of target (TI, T2, T3 or T4) was added to each reaction before incubation at 45° C for 6.5 min before running 53.5μΙ of the 60μΙ reaction on the lateral flow strip. Prior to application of the reaction to the lateral flow strip, 1.5pmoles of the second oligonucleotide probe and 2pg of carbon adsorbed to biotin-binding protein were deposited on the conjugate pad and allowed to dry for 5 min. Figure 8 presents a photograph of the nucleic acid lateral flow strips obtained in the experiment. The strip obtained with the TI target shows a clear black line corresponding to the detected species attached to the carbon attached to the solid material of the nitrocellulose and evidencing that the assay developed in this example including the oligonucleotide primers and probes works correctly and has the potential to fast and sensitive detection. The reactions performed with targets T2 and T3 did not reveal any carbon corresponding to the positive signal, evidencing that both the four mismatches as well as the deletion of four bases eliminates the ability of the second oligonucleotide to hybridize effectively to the predetector species produced in the reaction. A very weak signal was observed in the strip produced using T4 indicating that the presence of only two base mismatches leads to a substantial loss in the ability of the second oligonucleotide probe to successfully hybridize to the pre-detector species to produce the capable detecter species. of joining the line on the strip. Polyacrylamide gel electrophoresis using replicate reactions was performed to confirm that all reactions with all targets functioned correctly and produced a significant amount of amplification product. An expected size change was visible in the reaction performed with the four base truncated T3 site. This example demonstrates how the first and second oligonucleotide probes, an integral feature of the present invention, provide not only rapid and sensitive detection of the amplification product, but can also be used to provide additional target sequence-based verification of specificity. in the amplification product beyond that resulting from primer annealing alone. This powerful technique overcomes the known problems of the prior art resulting from non-target specific background amplification in certain assays that QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ result from ab initio synthesis or primer-primer ligation. It demonstrates that the method of the invention exhibits an increase in specificity compared to prior art methods, while retaining sensitive detection and resulting low-cost visualization. EXAMPLE 5 Carrying out the method where the portion that allows the 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 fluoside strip In the method of the invention, a number of different moieties can be used as the moiety for binding the second oligonucleotide probe to a solid material. This example demonstrates that the method can be performed where the portion that allows the 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. A second oligonucleotide probe was designed to comprise a 32 base sequence comprising a region of homology to at least one species in an amplification product and a 3' NHS Digoxigenin Ester modification that was added during synthesis. A sheep purified Fab fragment anti-digoxigenin antibody (Sigma-Aldrich) was immobilized onto a lateral flow strip of nucleic acid by spattering and air drying. The performance of the second oligonucleotide probe was demonstrated in an experiment where various levels of target (+++ = 1 pmol; ++ = 0.1 pmole; + = lOfmole; NTC = no target control) were added at 60μΙ of a planned reaction buffer containing the necessary reagents for detection using a nucleic acid-on-carbon lateral flow reaction, including 0.016 mgml·1 of carbon adsorbed to biotin-binding protein. The strip was prepared with 0.5pg of anti-digoxigenin Fab fragment spotted onto the strip in 0.2μΙ buffer containing 2.5mM Borate and 0.5% Tween 20. The solution was allowed to dry on the nitrocellulose membrane of the flow strip. side for 2h. Reactions were incubated at 45°C for 2 min to form the planned detectable species before the entire reaction mix from each reaction was applied to a lateral flow strip. Figure 9 presents a photograph of the lateral flow strips produced in the experiment. Black spots corresponding to carbon deposition on the lateral flow strip are visible at each level of the target and are not visible on the NTC indicating specific detection of the species detected. A combination of a biotin-based affinity interaction for the binding of the sensing (carbon) moiety and an antibody-based affinity interaction for the solid material binding moiety has been demonstrated. This example serves to demonstrate the QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ versatility of the method in terms of different approaches available for the attachment of the second oligonucleotide probe to a solid material. EXAMPLE 6 Embodiment of the method wherein the portion that allows the binding of the second oligonucleotide probe to a 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 attached to a solid material This example demonstrates carrying out the method wherein the portion that allows the second oligonucleotide probe to be attached to a solid material is a single-stranded oligonucleotide comprising four repeat copies of a three base DNA sequence motif. As described above, embodiments of the method that employ a single-stranded oligonucleotide as the detection portion of the second oligonucleotide probe present a straightforward and versatile aspect of the method, facilitating detection by lateral flow of nucleic acid and enabling easy detection. of multiple different target nucleic acids in the same sample. In addition, detection portions of the single-stranded oligonucleotide can be defined in advance and optimized for efficient hybridization on the strip to increase detection sensitivity and provide efficient scale-up fabrication of the nucleic acid lateral flow strip. In one aspect of the invention we observed a striking improvement in hybridization on the strip by using a single-stranded oligonucleotide detection portion comprised of multiple repeat copies of a DNA sequence motif. This example presents the results of multiple side-by-side experiments where the performance of an assay with the second oligonucleotide attached directly to the lateral flow strip is substantially increased by the use of a single-stranded detection portion comprising four repeat copies of a motif. of three base DNA sequence and wherein the reverse complement of said single-stranded oligonucleotide sequence is attached to the lateral flow strip. EXAMPLE 6.1: An assay was designed exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme. and is placed in contact with the sample simultaneously with the performance of step a). A first oligonucleotide probe with a total length of 25 bases was designed comprising in the 5' to 3' direction: A 5' biotin modification; a 7 base neutral region; the 5 bases of a restriction enzyme recognition site that is not a nicking enzyme; a region of 13 bases QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ capable of hybridizing to the first region of hybridization on the target comprising a phosphorothioate bond at the restriction enzyme cleavage site; and a 3' phosphate modification, where the biotin modification allows binding of the first oligonudeotide probe to a colorimetric dye, carbon nanoparticles, and the phosphate modification blocks its extension by translocation DNA polymerase. chain. Two alternative second oligonucleotide probes were designed to detect the same target species (I and II). The second T oligonudeotide probe was designed to contain in the 5' to 3' direction: 3 X repeats of a 14 base region capable of hybridizing to the reverse complement of the second hybridization sequence on the target; and a 9 X Thymidine base spacer. Stained nucleic acid lateral flow strips containing 30pmoles of the probe were prepared. The second alternate oligonudeotide probe 'H' was designed to contain in the 5'-3' direction: A 14 base region capable of hybridizing to the reverse complement of the second region of hybridization on the target; a neutral spacer of 5 X Thymidine bases; and a 12-base single-stranded oligonudeotide portion comprising 4 X repeats of a 3-base sequence motif that acts as the portion that allows the second oligonudeotide probe to be attached to a solid material. An additional single-stranded oligonudeotide was designed comprising in the 5' to 3' direction: an 11 X Thymidine base spacer; a 36 base region comprising a 12X repeat of the reverse complement to 3 base sequence motif that forms the portion that allows the second oligonudeotide II to be attached to a solid material. For the second oligonudeotide II probe nucleic acid lateral flow strips were prepared stained with 30pmoles of said additional single-stranded oligonudeotide. Reactions to test the performance of oligonucleotide probes I and II were performed containing: 0.5pmoles of the first oligonudeotide probe in 60μΙ of an appropriate buffer containing 0.016mgml-l carbon adsorbed to biotin-binding protein. The reactions for II were assembled in the same way but with the addition of 0.5pmole of the second oligonudeotide probe II. The target nucleic acid (a target single-stranded DNA representative of at least one species within the amplification product resulting from the designed assay reagents) was added at various levels (+++ = lpmol, ++ = 0.lpmol, NTC = without control of the target). The assembled reactions were incubated for 2 min at 45°C before the complete reaction mix was loaded onto the appropriate nucleic acid lateral flow strip. Figure 10A presents a photograph of the lateral flow strips obtained in the experiment, with the left panel presenting the results with the second oligonudeotide probe. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ I and the right panel presenting the results with the second oligonucleotide probe II. The black spots corresponding to the deposition of the detected species bound to the carbon were visualized in the presence of the target. For the second oligonucleotide probe II comprising the repeat sequence motif a stronger signal was observed at all levels of the target. EXAMPLE 6.2: A separate assay was then designed for a completely different target nucleic acid to demonstrate the versatility of such method modalities and their broad applicability. Oligonucleotide probes were designed for the relevant target nucleic acid, single-stranded DNA, in a similar manner to that described in Example 6.1; again with two versions of the second oligonucleotide probe referred to as T and ΊΓ and various levels of the target (+++ = lpmol, ++ = O.lpmoles, + = 0.OOlpmoles). An even more remarkable effect was observed as presented in the photograph of the produced lateral flow strips shown in Figure 10B. At the two lower target levels tested the second oligonucleotide probe I produced no signal while the corresponding repeat sequence oligonucleotide probe II produced a clear positive signal indicated by the black spots of the deposited carbon. This example reveals a marked improvement over lateral flow hybridization-based detection employing a second oligonucleotide detection portion comprising repeat copies of a DNA sequence motif. It demonstrates that a 100-fold improvement in detection sensitivity based on the lateral flow of nucleic acid from the detected species can be obtained. Signal intensity is increased and signal develops more rapidly, demonstrating the potential for such embodiments of the invention to be readily applicable to applications involving rapid detection, such as by nucleic acid lateral flow. Furthermore, the potential for use of a single-stranded oligonucleotide as the detection portion attached to the second oligonucleotide probe is exemplified. EXAMPLE 7 Use of the method for the detection of an RNA virus in clinical specimens This example demonstrates carrying out the method for detecting an RNA virus in clinical specimens, using the embodiment of the method where the first oligonucleotide probe is brought into contact with the sample simultaneously with the carrying out of amplification step a) and the portion that allows binding of the second oligonucleotide probe to a solid material is a single-stranded oligonucleotide comprising four repeat copies of a three-base DNA sequence motif and the reverse complement of said single-stranded oligonucleotide sequence is bound to a solid material. In several investigations we have routinely detected very low copies of RNA targets, such as viral genomic extracts. For example, using extracts from QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ quantified viral genome We have used the method of the invention to detect less than 100 equivalent copies of a virus genome in a total time of less than 10 min, with an amplification step of less than 5 min . This remarkable speed and sensitivity demonstrate the potential of the method for application in the diagnostic field. As such, in this example, we have developed an assay to detect a pathogenic single-stranded RNA virus and demonstrate the performance of that assay using virus-infected clinical specimens. The first oligonucleotide primer with a total length of 25 nucleotide bases was designed to comprise in the 5' to 3' direction: An 8 base stabilization region synthesized to contain phosphorothioate linkages between each base; the 5 bases of a recognition site for a restriction enzyme that is not a nicking enzyme; and a 12 base hybridizing region comprising the reverse complementary sequence of the first hybridizing sequence in the target nucleic acid, designed to target a region within the single-stranded RNA virus genome. The second oligonucleotide primer was designed to contain the same stabilization region but without the phosphorothioate linkages and the same restriction enzyme recognition sequence, but with the 12 base hybridization region capable of hybridizing to the reverse complement of the second primer sequence. hybridization. In this example the first restriction enzyme and the second restriction enzyme are the same restriction enzyme. The first and second hybridizing sequences on the target nucleic acid are separated by 0 bases. The oligonucleotide primers were designed using the target nucleic acid so that the nucleotide base downstream of the cleavage site in the reverse complement of the primers is Adenosine so that alpha thiol dATP is used as the modified dNTP for use in method. A phosphorothioate modification is inserted by strand displacement DNA polymerase, or reverse transcriptase to block cleavage of said reverse complementary strand. The first oligonucleotide probe with a total length of 24 bases was designed to comprise in the 5' to 3' direction: A 5' biotin modification added during synthesis wherein said biotin modification allows the first oligonucleotide probe to bind to a colorimetric dye, carbon nanoparticles, a stabilization region of 8 bases; the 5 bases of the recognition sequence for a restriction enzyme that is not a nicking enzyme wherein the cleavage site for said restriction enzyme in the first oligonucleotide probe is protected by a phosphorothioate internudeotide bond added during synthesis; an 11 base region capable of hybridizing to at least one species in the amplification product; and a 3' phosphate modification that prevents extension by strand displacement DNA polymerase. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ The second oligonucleotide probe with a total length of 31 bases was designed to comprise in the 5' to 3' direction: a 14 base region capable of hybridizing to the second single-stranded detection sequence 3' of the first detection sequence single-stranded in said at least one species in the amplification product; a spacer comprising 5 X Thymidine bases; 4 X repeats of a three base DNA sequence motif, the reverse complement to which is immobilized on the lateral flow strip. The immobilized lateral flow printed oligonucleotide with a total length of 47 bases is designed to comprise: A neutral spacer comprising 11 X Thymidine bases; a 12 X repeat of a 3 base sequence motif, which is complementary to the 3 base sequence motif of the second oligonucleotide probe. A lateral flow control oligonucleotide with a length of 20 bases was designed to comprise in the 5' to 3' direction: a 5 X triplet repeat that is different from that in the second oligonucleotide probe; a neutral spacer comprising 5 X Thymidine bases and a 3' biotin molecule, added during synthesis. The control oligonucleotide binds to its reverse complement on the lateral flow strip to verify a successful carbon lateral flow procedure. The reactions were prepared with: 1.8pmoles of the first primer; 9.6pmoles of the second primer; 3.6pmoles of the first probe; lpmol from the second probe; 300μΜ of Sp-dATP-aS from Enzo Life Sciences; 60μΜ of each dTTP, dCTP and dGTP; 28U of restriction enzyme; 14U of a Bacillus strand displacement DNA polymerase; 35U of a viral reverse transcriptase enzyme; 3.5U of RNaseH and 3pg carbon adsorbed to biotin-binding protein. 5μΙ nasopharyngeal swab specimen collected from patients at a clinical site (source Discovery Life Sciences) that included 7 virus-positive and 6 virus-negative clinical specimens (verified by PCR assay). Reactions were performed in a 70µl volume in an appropriate reaction buffer. Reactions were incubated at 45°C for 4 min 30 sec before the entire reaction was loaded onto a nucleic acid lateral flow strip imprinted with approximately 50 pmol of the reverse complement to the 3 base triplet repeat portion of the second. oligonucleotide probe (bottom) and the reverse complement to the control oligonucleotide (top line). Figure 11 presents a photograph of the lateral flow strips obtained in the example embodiment. The arrows indicate the position where the reverse complement to the triplet repeat portion of the second oligonucleotide probe has been imprinted (+) and therefore where the positive signal appears, and the position of the reverse complement to the control oligonucleotide (CTL ) that verifies a successful run of lateral flow and therefore appears in both positive and negative assays. The upper panel (+ve) shows the results obtained with the virus-positive clinical samples and the lower panel (-ve) those with the virus-negative samples. A light black line indicates that the presence of target nucleic acid is present in each of the positive samples, QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ 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 the detected species, such as through ab initio synthesis or primer-primer ligation. No false negatives were observed, evidencing the robustness of the method and its sensitivity through the different levels of copy number of the target nucleic acid present within different clinical specimens. EXAMPLE 8 Carrying out the method at different temperatures The method of the invention can be carried out efficiently over a wide range of temperatures and does not require temperature cycling, nor any hot or warm start, preheating or a controlled temperature decrease. This example demonstrates the performance of a typical test over a range of different temperatures. By selecting enzymes with the desired optimum temperature, and using a phosphorothioate base that lowers the melting temperature of hybridization after incorporation, as the assay has been easily performed where amplification is performed over a surprisingly wide range of temperatures. and spanning a normally low temperature range. A separate experiment further demonstrates that assays performed using the method of the invention can be performed without a requirement to preheat the sample before the start of step a), and where no loss of performance is observed when the temperature is increased during performance. of the amplification in step a). EXAMPLE 8.1: An assay was designed exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme. and is placed in contact with the sample simultaneously with the performance of step a). A first primer was designed to contain in the 5' to 3' direction: a 7 base neutral region; the recognition site of a restriction enzyme; and, an 11 base region capable of hybridizing to the first hybridizing sequence in the target nucleic acid, a target DNA. A second primer was designed containing in the 5' to 3' direction: a 7 base neutral region; the recognition site for the same restriction enzyme as the first primer; and a 12 base region capable of hybridizing to the reverse complement of the second hybridizing sequence in the target nucleic acid. A first oligonucleotide probe with a total length of 21 bases was designed comprising in the 5' to 3' direction: a 5' biotin modification; a 6 base neutral region; the bases of the restriction enzyme recognition site containing a 2nd overlap mismatch; a 10 base region capable of hybridizing to the first region of hybridization in the QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ target comprising a G-clamp modification in the 6th opposition; and a 3' phosphate modification, where the biotin modification allows binding of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the phosphate modification blocks its extension by strand displacement DNA polymerase. . A second oligonucleotide probe was designed to contain in the 5' to 3' direction: an 11 base region capable of hybridizing to the reverse complement of the second hybridizing sequence on the target; a 4 X Thymidine base spacer and 12 bases comprising 4 X repeats of a 3 base sequence motif that act as the portion that allows attachment of the second oligonucleotide probe to a solid material. An additional single-stranded oligonucleotide was designed to comprise in the 5' to 3' direction: an 11 X Thymidine base spacer; a 33 base region comprising an 11X repeat of the reverse complement to 3 base sequence motif that forms the portion that allows attachment of the second oligonucleotide to a solid material. For the second oligonucleotide probe nucleic acid lateral flow strips were prepared stained with 30pmoles of said additional single-stranded oligonucleotide. Reactions were prepared in appropriate buffer containing: 1.5pmoles of the first primer; 1.Opmol of the second primer; lpmol of the first oligonucleotide probe; 60μΜ of Sp-dATP-a-S from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; and, various levels of target DNA (++ = lamol, + = lOzmol, NTC = no target control). The assembled reactions were incubated for 2 min at target temperature (I = 37°C; II = 45°C, III = 50°C and IV = 55°C) before being initiated by the final addition of 5U of the restriction enzyme and 5U of a Bacillus strand displacement DNA polymerase to a final reaction volume of 25μΙ. Reactions were then incubated for 5min (TI) or 8min (T2) at the temperature of the relevant target. After incubation, each reaction was transferred to 75μΙ buffer containing 1.5pmoles of the second oligonucleotide probe and 8pg of carbon adsorbed to biotin-binding protein before application to the sample pad of the lateral flow strip. nucleic acid. Figure 12A presents photographs of the lateral flow strips obtained in the experiment at each target level, temperature, and time point. The light black lines observed correspond to the deposition of the detected species bound to the carbon produced in the presence of the target. At all temperatures a very strong signal appeared in the presence of target at both target levels within 8 min demonstrating the wide temperature range of efficient amplification of the method. No non-specific amplification was observed in the NTC samples. Strong amplification was also observed after only 5 min at 45°C and 50°C indicating that the optimal temperature for this assay is probably between 40°C and 50°C. EXAMPLE 8.2: A second trial was designed exploiting the modality of the method QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ wherein the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and is placed in contact with the sample simultaneously to the performance of step a). Both of the first and second primers were designed to contain in the 5' to 3' direction: a 6 base neutral region; the recognition site of a restriction enzyme; and a 12 base region of hybridization to the target nucleic acid. The primers were designed so that the first and second hybridizing sequences on the target are 10 bases apart. A first oligonucleotide probe was designed with a total length of 23 bases comprising in the 5' to 3' direction: a 5' biotin modification; a 6 base neutral region; the bases of the restriction enzyme recognition site containing a mismatch at the 4th position; a 12 base region capable of hybridizing to the first region of hybridization on the target; and a 3' phosphate modification, where the biotin modification allows binding of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the phosphate modification blocks its extension by strand displacement DNA polymerase. . A second oligonucleotide probe was designed to contain in the 5' to 3' direction: a 13 base region capable of hybridizing to 3 bases of the reverse complement of the second hybridizing sequence on the target and the 10 base gap between the first and second hybridization sequences; a 3 X Thymidine base spacer and 12 bases comprising 4 X repeats of a 3 base sequence motif that acts as the portion that allows the second oligonucleotide probe to be attached to a solid material. An additional single-stranded oligonucleotide was designed to comprise in the 5' to 3' direction: an 11 X Thymidine base spacer; a 36 base region comprising a 12X repeat of the reverse complement to 3 base sequence motif that forms the portion that allows attachment of the second oligonucleotide to a solid material. For the second oligonucleotide probe nucleic acid lateral flow strips were prepared stained with 30pmoles of said additional single-stranded oligonucleotide. Reactions were prepared in appropriate buffer containing: 6pmoles of oligonucleotide first primer; 8pmoles of the second oligonucleotide primer; 6pmoles of the first oligonucleotide probe; 60μΜ of Sp-dATP-a-S from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; 60pg carbon adsorbed to biotin binding protein; and, where applicable, the target. The assembled reactions were incubated for 2 min at the starting temperature (I = 15°C; II = 45°C) before the reactions were started by the final addition of 20U of restriction enzyme, 20U of a Bacillus strand displacement DNA and 40U reverse transcriptase to a final reaction volume of 100μΙ. After the addition of the enzyme, the reactions with the starting temperature of 15°C were immediately transferred to 45°C along with the other reactions. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ Then the reactions were incubated for 6 min at 45°C. After incubation, each reaction was transferred to the sample pad of a nucleic acid lateral flow strip, which sample pad contained 3pmoles of the second oligonucleotide probe. Figure 12B presents photographs of the lateral flow strips obtained in the experiment at each of the incubation temperature conditions. The light black lines observed correspond to the deposition of the detected species bound to the carbon produced in the presence of the target. No difference was observed in the reaction where the temperature was increased from 15°C to 45°C during amplification step a). The same remarkable rate of amplification occurred as in the pre-heated reaction, and no non-specific amplification was observed in the NTC sample. This Example 8 demonstrates that the method of the invention can be used to easily develop assays with a lower optimum temperature profile compared to known methods, and which can be exploited for sensitive detection over an unusually wide temperature range. It also demonstrates that the method of the invention can be performed without preheating where the temperature is increased during the performance of step a). Such features are highly attractive for use of the method in a low-cost diagnostic device, where high temperatures and precisely controlled heating impose complex physical constraints that increase the cost of goods for such a device to a point where a diagnostic device a single use or free instrument is not commercially viable. Furthermore, by avoiding the requirement of known methods to preheat the sample before the start of amplification, the method of the invention can be performed with fewer user steps and a simpler sequence of operations, thus increasing the usability of such a device. diagnostic device and decreasing the total time to result. EXAMPLE 9 Carrying out the method in which the target nucleic acid is derived from double-stranded DNA by strand invasion This example demonstrates the use of the method where the single-stranded target nucleic acid is a single-stranded site within double-stranded DNA that is detected without any requirement for specific action to separate the DNA strands, such as temperature denaturation, skipping primers, or use of an additional enzyme (eg helicase or recombinase). The ability to use the method of the invention easily for the detection of both single-stranded RNA and double-stranded DNA targets makes it highly versatile for use in diagnostic applications, with no additional user steps, components, or physical requirements imposed on the device used to perform the method. QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ EXAMPLE 9.1: An assay for a protein-coding region within the double-stranded DNA genome of a viral target was developed. It is possible to use either the double-stranded genome or the mRNA transcript as a biomarker for the presence of the virus in clinical diagnosis. The assay was designed exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and is placed in contact with the sample simultaneously to the performance of step a). The design of the oligonucleotide primers and oligonucleotide probes was performed following a similar approach to that described in other examples, without a gap between the first and second hybridization sequences in the target nucleic acid. Reactions were prepared in appropriate buffer containing: 4pmoles of oligonucleotide first primer; 2pmole of the second oligonucleotide primer; 2pmoles of the first oligonucleotide probe; 60μΜ of Sp-dATP-a-S from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; 60pg carbon adsorbed to biotin-binding protein; and either target (I) double-stranded DNA or target (II) single-stranded RNA or no target. The assembled reactions were incubated for 2 min at 45°C before being initiated by the final addition of 20U of the restriction enzyme, 20U of a BaciIIus strand displacement DNA polymerase, and 25U of reverse transcriptase to a final volume of ΙΟΟμΙ reaction. After the addition of the enzyme, the reactions were incubated at 45°C for 7 min. After incubation, 1.Spmoles 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 site was also added to all samples. Figure 13A presents photographs of the lateral flow strips obtained in the experiment with each target. The light black lines observed correspond to the deposition of the carbon-bound detecting species produced in the presence of the target, with a weaker signal corresponding to the lower level of the target (+) than the higher level of the target (++). . No difference in amplification rate was observed between single-stranded RNA and double-stranded DNA targets. EXAMPLE 9.2: An assay was designed for a single-stranded target nucleic acid within the c.2.5 megabase double-stranded DNA genome of a bacterial pathogen. The assay was designed exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and is placed in contact with the sample simultaneously to the performance of step a). The design of the oligonucleotide primers and oligonucleotide probes was performed following a similar approach to that described in other examples, with a 4 base gap between the first and second hybridization sequences in the target nucleic acid. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ Reactions were prepared in appropriate buffer containing 2pmoles of the first oligonucleotide primer and 0.5pmoles of the second oligonucleotide primer. Due to the use of a double-stranded DNA target, two single-stranded target nucleic acids are in fact added at the same time and it is assumed that a second reciprocal process also occurs, where 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 impact on the performance of the method. 2pmoles of the first oligonucleotide probe; 60μΜ of Sp-dATP-a-S from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; 15pg carbon adsorbed to biotin binding protein; and genome extract of the bacterium containing the target at various levels (++ = lamol; + = lOzmoles; NTC = no target control). An additional specificity control reaction containing 1 mol of E. coH genome extract was also performed. The assembled reactions were incubated for 3 min at 45°C before being initiated by the final addition of 4U of the restriction enzyme and 2U of a Bacillus strand displacement DNA polymerase to a final reaction volume of 25μΙ. After the addition of the enzyme, the reactions were incubated at 45°C for 6 min. After incubation, 75µΙ of buffer containing 3pmoles of the second oligonucleotide probe was added to each reaction and the entire volume was then transferred to the sample pad of a nucleic acid lateral flow strip. Figure 13B presents photographs of the lateral flow strips obtained in the experiment with each target. Light black lines corresponding to the deposition of the carbon-bound detecting species produced in the presence of the target are observed at both levels of the target tested. No non-specific signal was observed in the no-target control or in the presence of E. coH genomic DNA demonstrating that the method can be employed for the specific detection of a complex double-stranded DNA genome at a clinically relevant copy number within only 6 minutes This example demonstrates that the method of the invention can be readily used for the detection of single-stranded nucleic acid targets within double-stranded DNA. Notably, a similar amplification rate is observed for the detection of single-stranded RNA target and the same target sequence within double-stranded DNA, without any requirement for specific action such as temperature denaturation to separate the DNA duplex. Instead of the single-stranded site being sufficiently exposed for hybridization and extension of the first oligonucleotide primer to initiate the strand invasion method wherein the transient opening of one or more DNA base pairs within the double-stranded DNA occurs sufficiently to allow the annealing and extension of the 3' hydroxyl of the first oligonucleotide primer. This is in contrast to known methods such as SDA where thermal denaturation and jumping primers are used in assays for double-stranded nucleic acids. usability QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ of the invention method for easily detecting targets within double-stranded DNA as well as those within single-stranded DNA and single-stranded RNA make it highly versatile for use in diagnostic applications, such as in the detection of bacterial pathogens , fungal and viral that have a double-stranded DNA genome. The fact that the method does not require complex additional user steps, enzymes, components, or physical constraints to detect organisms with double-stranded DNA genomes means that it is particularly highly suitable for testing in a simple, low-cost diagnostic device. For example, the requirement for thermal denaturation prior to amplification performance reported for known methods could necessitate additional expensive components and increase the cost of goods for such a device and the total time to a result, meaning it would not be feasible. a single-use or self-contained instrument-free device. EXAMPLE 10 Comparative performance of the method of the invention against known methods This example presents a comparative evaluation of the method of the invention against the known method described in WO2014 / 164479 for the detection of a viral target. The known method is fundamentally different from the method of the invention in that it requires nicking enzymes and does not require the use of one or more modified dNTPs. The method of the invention is shown to have vastly superior sensitivity and specificity. For this comparative evaluation an assay for a viral target with a single stranded RNA genome was first developed using the method of the invention. Said assay was designed exploiting the embodiment of the method where the first oligonucleotide probe is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme and put into contact with the sample simultaneously to the performance of step a). The design of the oligonucleotide primers and oligonucleotide probes was performed following a similar approach to that described in other examples, with a 6 base gap between the first and second hybridization sequences in the target nucleic acid. For the assay using the known method, similar primers were designed containing the same 6 base neutral region at the 5' ends and the same hybridizing regions at the 3' end as the equivalent primers used in the method of the invention. In this way consistency was ensured as much as possible between the two trials for an accurate comparison of methods. However, the bases of the restriction enzyme recognition site were replaced with those of the exemplary nicking enzyme reported in WO2014 / 164479, Nt.BbvCI (see Example 5 on p.20-21). QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ EXAMPLE 10.1: In the first case, the reactions for each method were performed using equal primer ratios. For the method of the invention, reactions were prepared in appropriate buffer containing: 2pmoles of the oligonudeotide first primer; 2pmoles of the second oligonudeotide primer; 1.6pmoles of the first oligonudeotide probe; 60μΜ of Sp-dATP-a-S from Enzo Life Sciences; 60μΜ each of dTTP, dCTP and dGTP; and extract of viral genomic RNA at various levels as target (+++ = 10zmoles; ++ = 100 copies; + = 10 copies; NTC = no target control). The assembled reactions were preincubated for 5 min at room conditions (c.20°C) before the reactions were initiated by the addition of 5U of the restriction enzyme, 5U of a Bacillus strand displacement DNA polymerase, and 10U of of reverse transcriptase in a final reaction volume of 25μΙ. After the addition of the enzyme, the reactions were incubated at 45°C for 8 min (TI) or 15 min (T2). After incubation, 60pg of biotin-binding protein-adsorbed carbon in 75μΙ buffer was added to each reaction and the entire 100μΙ volume was transferred to a nucleic acid lateral flow strip containing 1.5pmoles of the second probe. oligonudeotide on the sample pad. For the known method, reactions were prepared in appropriate buffer containing: 6.25pmoles of oligonudeotide first primer; 6.25pmoles of the second oligonudeotide primer; 200μΜ each of dATP, dTTP, dCTP and dGTP; and viral genomic RN extract at various levels as target (+++ = 10zmoles; ++ = 100 copies; + = 10 copies; NTC = no target control). The assembled reactions were preincubated for 5 min at room conditions (c.20°C) before the reactions were initiated by the addition of 4U of Nt.BbvCI, 20U of Bst large fragment DNA polymerase and 10U of Bst reverse transcriptase. M-MuLV in a final reaction volume of 25μΙ. After the addition of the enzyme, the reactions were incubated at 45°C for 8 min (TI) and 15 min (T2). After incubation, 75μΙ of buffer containing 60pg carbon adsorbed to biotin-binding protein and 5pmoles of the first oligonudeotide probe were added to each reaction and the entire 100μI volume was transferred to a nucleic acid lateral flow strip. containing 5pmoles of the second oligonudeotide probe in the sample pad. Figure 14A presents photographs of the lateral flow strips obtained in the experiment with the method of the invention (I) and with the known method (II), at the various target levels and time points indicated. The light black lines observed correspond to the deposition of the carbon-bound detector species produced in the presence of the target. Many trials were required before it was possible to observe any signal at all using the known method and it was necessary to use a particular combination of enzymes and regulator and significantly higher levels of primers, dNTPs and enzymes. With the method of the invention (I), even at the shortest time point after only 8 min without a pre-heat it was possible to clearly see the detector species produced even at the lowest target level of only 10 copies. QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ of the target. Even after efforts to optimize the known method which might not have been obvious to the skilled person, only a weak signal was observed at the highest level of the target (+++ = lOzmoles) and at the longest time point ( 15 minutes). EXAMPLE 10.2: After extensive, non-obvious additional attempts, it was possible to increase the performance of the known method, but only by using a 2:1 ratio of the first and second oligonucleotide primers, with a very high concentration of the first primer, as is described in this Example 10.2. The method of the invention was carried out again as described in Example 10.1. For the known method, reactions were performed as described in Example 10.1 except that the level of the first oligonucleotide primer was increased to 12.5pmoles. In each case the following levels of the target were used: +++ = lzmol; ++ = 100 copies; + = 10 copies; NTC = no target control. Figure 14B presents photographs of the lateral flow strips obtained in the experiment with the method of the invention (I) and with the known method (II), at the various target levels and time points indicated. The light black lines observed correspond to the deposition of the carbon-bound detecting species produced in the presence of the target. Again, the method of the invention (I) demonstrated remarkable speed with the signal visible even at the shortest time point and the lowest target level of only 10 target copies. With the known method only a weak signal was observed at the highest level of the target (+++ = lzmol) and a very weak signal was visible in the 100 copy sample at the longest time point (15 min). However, a weak signal was also observed in the NTC strip which may correspond to a non-specific product as a result of the very high levels of oligonucleotide primer and enzyme levels required to make the method work to a minimum. These data are consistent with the data in WO2014 / 164479 where an incubation time of 30 min was reported. The requirement to add unusually high primer levels to speed up the amplification performed using this known method could greatly limit its potential application for the detection of two or more different targets in the same sample, as there would be very limited scope to further increase. the total level of the primer without exacerbating the problem with non-specific products. This Example 14 demonstrates the marked superiority of the method of the invention over the known method described in WO2014 / 164479 with the amplification performed much faster, with higher sensitivity and with a resulting clearer signal produced. In just 8 min without pre-incubation the method of the invention produced a stronger signal with only 100 copies of the target than the known method was capable of in 15 min at the highest target level with 60X the target level. . The advantages of the method of the invention over this known method arise from its requirement for a different class of enzyme, which are restriction enzymes than are not nicking enzymes, and from its requirement for the use of one or more modified dNTPs, such as a base of QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ phosphorothioate that increases the sensitivity and specificity of amplification. Furthermore, the embodiment of the method wherein one of the first and second oligonucleotide probes is blocked at the 3' end of the extension by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzyme. and contacted with the sample simultaneously with the performance of step a), enables efficient coupling of amplification to signal detection and facilitates an increase in specificity derived from efficient sequence-based hybridization during formation of samples. the detector species. These advantages make the method of the invention ideally suitable for exploitation in the diagnostic field and for the development of simple, ultra-fast, user-centered, low-cost devices, such as an instrument-free molecular diagnostic test device. single use only. Throughout the specification and claims that follow, unless the context otherwise requires, the word comprise and variations such as comprise and comprise shall be understood to imply the inclusion of a declared integer, step, group of integers, or step group, but not to the exclusion of any other integer, step, integer group, or step group. Additional aspects of the invention include those listed below: 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample comprising: a) Put the sample in contact with: Yo. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and a cleavage site and a region that is capable of hybridizing to a first hybridization sequence to the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of reverse complement hybridization of a second hybridizing sequence downstream of the first hybridizing sequence in the target nucleic acid; ii. a strand displacement DNA polymerase; iii. the dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but is capable of annealing the recognition sequence of the first primer and cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; and I saw. a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product from step a) with: Yo. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is linked to a portion that allows its detection; and ii. a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence in said at least one species within the amplification product and that is attached to a solid material or to a portion that allows attachment to a solid material; wherein hybridization of the first and second probes to said at least one species within the amplification product produces a detectable species; and c) detecting the presence of the detect species produced in step b) wherein the presence of the detect species indicates the presence of the target nucleic acid in said sample. 2. A method according to aspect 1 wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second enzyme of restriction. 3. A method according to aspect 2 wherein the one oligonucleotide probe is rendered incapable of being cleaved by either the first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more further modifications such as a phosphorothioate bond. 4. A method according to aspect 2 or 3 wherein the oligonucleotide probe is contacted with the sample simultaneously for carrying out step a). QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ 5. A method according to any of the above aspects wherein the sample is further contacted in step a) with: (a) a third oligonucleotide primer, said third primer comprising in the 5' to 3' direction a strand of the recognition sequence and cleavage site for the first restriction enzyme and a region which is capable of hybridizing to the first hybridizing sequence in the target nucleic acid and wherein said third primer is blocked at the 3' end of the extension by DNA polymerase; and / or (B) a fourth oligonucleotide primer, said fourth primer comprising in the 5' to 3' direction a strand of the recognition sequence and cleavage site for the second restriction enzyme and a region that is capable of hybridizing to the reverse complement of the second hybridization sequence into the target sequence and wherein said fourth primer is blocked at the 3' end of the extension by DNA polymerase. 6. A method according to aspect 5 wherein when the third oligonucleotide primer is present, the first oligonucleotide primer is provided in excess and when the fourth oligonucleotide primer is present, the second oligonucleotide primer is provided in excess. 7. A method according to any of the above aspects wherein the one or more modified dNTPs is a modified alpha thiol dNTP. 8. A method according to any of the above aspects wherein the first and second restriction enzymes are the same restriction enzyme. 9. A method according to any of the above aspects where two or more of steps a), b) and c) are performed simultaneously. 10. A method according to any of the above aspects wherein step (a) is carried out at a temperature of not more than 50°C. 11. A method according to any of the above aspects wherein the portion that allows detection of the first oligonucleotide probe is a fluorometric or colorimetric dye or a portion that is capable of binding to a fluorometric or colorimetric dye such as biotin . 12. A method according to any of the above aspects in which the species to be detected is detected by a change in the electrical signal. 13. A method according to any of the above aspects wherein the detection-enabling portion of the first oligonucleotide probe is an enzyme that produces a detectable signal, such as a colorimetric or fluorometric signal, after contact with a substrate. 14. A method according to any of the above aspects wherein the portion that allows the attachment of the second oligonucleotide probe to a solid material is a QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ single-stranded oligonucleotide. 15. A method according to aspect 14 wherein the sequence of the single-stranded oligonucleotide portion comprises three or more repeat copies of a 2 to 4 base DNA sequence motif. 16. A method according to any of the above aspects wherein in step c) the presence of the species to be detected is detected by lateral flow of nucleic acid. 17. A method according to aspect 16 wherein the lateral flow of nucleic acid uses one or more nucleic acids that are capable of sequence-specific hybridization to the moiety that allows binding of the second oligonucleotide probe to a solid material. 18. A method according to any of the above aspects wherein step c) produces a colorimetric or electrochemical signal using carbon or gold, preferably carbon. 19. A method according to any of the above aspects wherein the first and / or second oligonucleotide primers comprise a stabilizing sequence at the 5' end, for example 5 bases in length downstream of the recognition sequence of restriction enzyme and cleavage site. 20. A method according to any of the above aspects wherein the hybridizing region of the first and / or second oligonucleotide primers is between 9 and 16 bases in length. 21. A method according to any of the above aspects wherein one of the first and second oligonucleotide primers is provided in excess of the other. 22. A method according to any of the above aspects wherein the first and second hybridizing sequences in the target nucleic acid are separated by 0 to 6 bases. 23. A method according to any of the above aspects wherein the first and second hybridizing sequences in the target nucleic acid are separated by 3 to 6 bases. 24. A method according to any of the previous aspects wherein in step b) any of the first or second single-stranded detection sequence in the at least one species within the amplification product includes the sequence corresponding to the 3 to 6 bases defined in claim 23. 25. A method according to any of the above aspects wherein the level of the target nucleic acid in said sample is quantified in step c). 26. A method according to any of the above aspects wherein the target nucleic acid is single-stranded RNA, including RNA-derived single-stranded RNA QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ double-stranded 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. 27. A method according to aspect 26 wherein said single-stranded DNA is derived from double-stranded DNA by the use of a nuclease, such as a restriction endonuclease or exonuclease III or derived from single-stranded RNA by the use of reverse transcriptase. 28. A method according to any of the above aspects wherein the presence of two or more different target nucleic acids of defined sequence in the same sample is detected. 29. A method according to any of the preceding aspects wherein the sample is a biological sample, such as a nasal or nasopharyngeal swab or aspirate, blood or blood-derived sample, or urine. 30. A method according to any of the preceding aspects in which the target nucleic acid is viral or derived from viral nucleic acid material, is bacterial or derived from bacterial nucleic acid material, is circulating cell-free DNA released from cancer cells or fetal cells, it is microRNA or microRNA derivative. 31. A method according to any of the above aspects wherein the target nucleic acid contains a site of epigenetic modification, such as methylation. 32. A method according to any of the preceding aspects wherein the detection of the target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or a disease state. 33. A method according to aspect 32 wherein said disease is a disease, including but not limited to HIV, influenza, RSV, Rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile virus, Epstein-Barr, malaria, plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox. 34. A method according to 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, kidney cancer, stomach cancer or melanoma. 35. A method according to any of the above aspects wherein the detection of said target nucleic acid is used for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics or pharmacokinetics. 36. A method according to any of the preceding aspects wherein the sample is a human sample, forensic sample, agricultural sample, veterinary sample, environmental sample, or biodefense sample. 37. A kit comprising: QbRnnn / 1 7Π7 / Ε / ΥΙΛΙ a) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridizing to a first hybridization sequence to a single-stranded target nucleic acid of defined sequence, and said second primer comprises in the 5' to 3' direction a restriction enzyme recognition sequence and cleavage site and a region that is 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 that is not a nicking enzyme and is capable of recognizing the recognition sequence of and cleaving the first primer cleavage site and a second restriction enzyme that is not a nicking enzyme and is capable of recognizing the sequence recognizing and cleaving the second primer cleavage site; c) a strand displacement DNA polymerase; d) dNTPs; e) one or more modified dNTPs; f) a first oligonucleotide probe that has a part of complementarity to the hybridization region of one of the first and second oligonucleotide primers and is linked to a part that allows its detection; and g) a second oligonucleotide probe that has a part of complementarity to the reverse complement of the hybridization region of the other of the first and second oligonucleotide primers and is bound to a solid material or to a portion that allows its binding to a solid material. 38. A kit according to aspect 37 further comprising means for detecting the presence of the species to be detected. 39. A 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 of items 2, 3, 7, 8, 11, 13 to 17,19, 20 or 22 to 24. 40. A kit according to any of aspects 37 to 39 further comprising third and / or fourth oligonucleotide primers as defined in aspect 5 or 6. 41. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample comprising: a) put the sample in contact with: QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ Yo. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and a cleavage site and a region that is capable of hybridizing to a first hybridization sequence to the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of reverse complement hybridization of a second hybridizing sequence downstream of the first hybridizing sequence in the target nucleic acid; ii. a strand displacement DNA polymerase; iii. the dNTPs; iv. one or more modified dNTPs; v. a first restriction enzyme that is not a nicking enzyme but is capable of annealing the recognition sequence of the first primer and cleaving only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; and I saw. a second restriction enzyme that is not a nicking enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product from step a) with: Yo. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is linked to a portion that allows its detection; and ii. a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence upstream or downstream of QHRnnn / 1 7Π7 / Ε / ΥΙΛΙ the first single-stranded detection sequence in said at least one species within the amplification product and which is bound to a solid material or to a portion allowing its binding to a solid material; wherein one of the first and second oligonucleotide probes is blocked from extension at the 3' end by DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, and where hybridization of the first and second probes to said at least one species within the amplification product produces a detectable species; and c) detecting the presence of the detect species produced in step b) wherein the presence of the detect species indicates the presence of the target nucleic acid in said sample; and wherein any of the first or second oligonucleotide probes defined in step b) is placed in contact with the sample simultaneously with the performance of step a). All patents and patent applications referenced herein are incorporated by reference in their entirety.

Claims

CLAIMS 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, characterized in that it comprises: a) contacting the sample with: i. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and a cleavage site and a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, and said second primer comprises in the 5' to 3' direction a strand of a restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the reverse complement of a second hybridization sequence in the 5' direction of the first hybridization sequence in the target nucleic acid; ii. a strand-displacement DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v.a first restriction enzyme that is not a nick enzyme but is able to annotate the recognition sequence of the first primer and cleave only the first primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, the cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTPs; and vi.a second restriction enzyme that is not a nick enzyme but is capable of recognizing the recognition sequence of the second primer and of cleaving only the second primer strand from the cleavage site when said recognition sequence and cleavage site are double-stranded, the cleavage of the reverse complementary strand is blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTPs; to produce, without temperature cycling, in the presence of said target nucleic acid, the amplification product; b) contacting the amplification product of step a) with: i. a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species within the amplification product and that is attached to a portion that allows its detection; and ii.a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence in the 5' or 3' direction of the first single-stranded detection sequence on the same strand of said at least one species within the amplification product and that is bound to a solid material or a portion that allows its binding to a solid material; wherein hybridization of the first and second probes to said at least one species within the amplification product produces a detector species; and c) detecting the presence of the detector species produced in step b), wherein the presence of the detector species indicates the presence of the target nucleic acid in said sample.

2. The method according to claim 1, further characterized in that one of the first and second oligonucleotide probes has its extension at the 3' end blocked by the DNA polymerase and is not capable of being cleaved by either of the first or second restriction enzymes.

3. The method according to claim 2, further characterized in that the blocked oligonucleotide probe becomes unable to be cleaved by either the first or second restriction enzymes due to the presence of one or more sequence mismatches and / or one or more modifications such as a phosphorothioate bond.

4. The method according to claim 2 or 3, further characterized in that the blocked oligonucleotide probe is brought into contact with the sample simultaneously with the performance of step a).

5. The method according to any of claims 2 to 4, further characterized in that the locked oligonucleotide probe comprises an additional region such that the 3' end of the species within the amplification product to which the locked oligonucleotide probe is hybridized can be extended by the strand displacement DNA polymerase.

6. The method according to any of the preceding claims, further characterized in that the sample is additionally brought into contact in step a) with: (a) a third oligonucleotide primer, said third primer comprising in the 5' to 3' direction a strand of the recognition sequence and cleavage site for the first restriction enzyme and a region capable of hybridizing to the first hybridization sequence in the target nucleic acid, and wherein said third primer is blocked at the 3' end of the extension by the DNA polymerase;and / or (B) a fourth oligonucleotide primer, said fourth primer comprising in the 5' to 3' direction a strand of the recognition sequence and cleavage site for the second restriction enzyme and a region that is capable of hybridizing to the reverse complement of the second hybridization sequence in the target sequence and wherein said fourth primer is blocked at the 3' end of the extension by the DNA polymerase.

7. The method according to claim 6, further characterized in that when the third oligonucleotide primer is present, the first oligonucleotide primer is provided in excess, and when the fourth oligonucleotide primer is present, the second oligonucleotide primer is provided in excess.

8. The method according to any of the preceding claims, further characterized in that the one or more modified dNTPs is a modified alpha thiol dNTP. QbRnnn / 1 7P7 / E / YILI 9. The method according to any of the preceding claims, further characterized in that the first and second restriction enzymes are the same restriction enzyme.

10. The method in accordance with any of the preceding claims, further characterized in that two or more of steps a), b) and c) are performed simultaneously.

11. The method in accordance with any of the preceding claims, further characterized in that step a) is performed at a temperature of no more than 50°C.

12. The method in accordance with any of the preceding claims, further characterized in that the temperature is increased during the performance of step a), such as an increase from an initial ambient temperature, for example in the range of 15-30°C, to a temperature in the range of 40-50°C.

13. The method according to any of the preceding claims, further characterized in that the portion enabling the detection of the first oligonucleotide probe is a fluorometric or colorimetric dye or a portion capable of binding to a fluorometric or colorimetric dye such as biotin.

14. The method in accordance with any of the preceding claims, further characterized in that the species to be detected is detected by a change in the electrical signal.

15. The method according to any of the preceding claims, further characterized in that the portion enabling the detection of the first oligonucleotide probe is an enzyme that produces a detectable signal, such as a colorimetric or fluorometric signal, after contact with a substrate.

16. The method according to any of the preceding claims, further characterized in that the portion that allows the second oligonucleotide probe to bind to a solid material is a single-stranded oligonucleotide.

17. The method according to claim 16, further characterized in that the sequence of the single-stranded oligonucleotide portion comprises three or more repeated copies of a DNA sequence motif of 2 to 4 bases.

18. The method in accordance with any of the preceding claims, further characterized in that in step c) the presence of the species to be detected is detected by the lateral flow of nucleic acid.

19. The method according to claim 18, further characterized in that the nucleic acid lateral flow utilizes one or more nucleic acids that are capable of sequence-specific hybridization to the portion that allows the second oligonucleotide probe to bind to a solid material.

20. The method in accordance with any of the preceding claims, QbRnnn / 1 7P7 / E / YILI, further characterized in that in step c) it produces a colorimetric or electrochemical signal using carbon or gold, preferably carbon.

21. The method according to any of the preceding claims, further characterized in that the first and / or second oligonucleotide primers comprise a stabilizing sequence in the 5' direction of the restriction enzyme recognition sequence and cleavage site, for example 5 or 6 bases in length.

22. The method according to any of the preceding claims, further characterized in that the hybridization region of the first and / or second oligonucleotide primers is between 9 and 16 bases in length.

23. The method in accordance with any of the preceding claims, further characterized in that one of the first and second oligonucleotide primers is provided in excess of the other.

24. The method according to any of the preceding claims, further characterized in that the first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 bases.

25. The method according to any of the preceding claims, further characterized in that the first and second hybridization sequences in the target nucleic acid are separated by 3 to 15 bases.

26. The method according to any of the preceding claims, further characterized in that in step b) any of the first or second single-stranded detection sequence in the at least one species within the amplification product includes at least 3 bases from the sequence corresponding to the 3 to 15 bases defined in claim 24.

27. The method in accordance with any of the preceding claims, further characterized in that the level of the target nucleic acid in said sample is quantified in step c).

28. The method according to any of the preceding claims, further characterized in that 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.

29. The method according to claim 28, further characterized in that the target single-stranded nucleic acid is single-stranded DNA derived from double-stranded DNA by strand invasion.

30. The method according to claim 28, further characterized in that said single-stranded DNA is derived from double-stranded DNA by the use of a nuclease, such as QbRnnn / 1 7P7 / E / YILI a restriction endonuclease or exonuclease III or derived from single-stranded RNA by the use of reverse transcriptase.

31. The method in accordance with any of the preceding claims, further characterized in that the presence of two or more different target nucleic acids of defined sequence is detected in the same sample.

32. The method according to any of the preceding claims, further characterized in that the sample is a biological sample, such as a nasal or nasopharyngeal swab or aspirate, blood or a blood-derived sample, or urine.

33. The method according to any of the preceding claims, further characterized in that the target nucleic acid is viral or derived from viral nucleic acid material, is bacterial or derived from bacterial nucleic acid material, is circulating DNA, cell-free DNA released from cancer cells or fetal cells, is microRNA or derived from microRNA.

34. The method according to any of the preceding claims, further characterized in that the detection of the target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or disease state.

35. The method according to claim 34, further characterized in that 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.

36. The method according to claim 34, further characterized in that said disease is 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, kidney cancer, stomach cancer or melanoma.

37. The method in accordance with any of the preceding claims, further characterized in that the detection of said target nucleic acid is used for human genetic testing, prenatal testing, blood contamination testing, pharmacogenomics or pharmacokinetics.

38. The method in accordance with any of the preceding claims, further characterized in that the sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, and an environmental sample or a biodefense sample.

39. A kit characterized in that it comprises: a) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 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 said second primer comprises in the 5' to 3' direction a QbRnnn / 1 7P7 / E / YILI restriction enzyme recognition sequence and cleavage site and a region capable of hybridizing to the inverse complement of a second hybridization sequence in the 5' direction of the first hybridization sequence in the target nucleic acid;b) a first restriction enzyme that is not a nick enzyme and is capable of recognizing the recognition sequence and dissociating the cleavage site of the first primer and a second restriction enzyme that is not a nick enzyme and is capable of recognizing the recognition sequence and dissociating the cleavage site of the second primer; c) a strand displacement DNA polymerase; d) the dNTPs; e) one or more modified dNTPs; f) a first oligonucleotide probe that is capable of hybridizing to a first single-stranded detection sequence in at least one species in the amplification product produced in the presence of said target nucleic acid and that is attached to a portion that allows its detection;(g) a second oligonucleotide probe that is capable of hybridizing to a second single-stranded detection sequence in the 5' or 3' direction of the first single-stranded detection sequence on the same strand of said at least one species in the amplification product and that is bound to a solid material or a portion that allows its binding to a solid material.

40. The kit according to claim 39 further characterized in that one of the first and second oligonucleotide probes has its extension at the 3' end blocked by DNA polymerase and is not capable of being cleaved by either of the first or second restriction enzymes, for example due to the presence of one or more sequence mismatches and / or one or more modifications such as a phosphorothioate bond.

41. The kit according to claim 39 or 40, further characterized in that one of the first and second oligonucleotide probes has 5 or more bases complementary to the hybridization region or the inverse complement of the hybridization region of the first or second primer.

42. The kit according to claim 41, further characterized in that the first oligonucleotide probe has 5 or more bases complementary to the hybridization region of one of the first and second oligonucleotide primers, and the second oligonucleotide probe has 5 or more bases complementary to the inverse complement of the hybridization region of the other of the first and second oligonucleotide primers.

43. The kit according to any of claims 39 to 42, further characterized in that it additionally comprises means for detecting the presence of a detector species produced in the presence of the target nucleic acid.

44. The kit according to any of claims 39 to 43, further characterized in that 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 any of the first or second single-stranded detection sequence in at least one species within the amplification product are as defined in any of claims 5, 8, 9, 13, 15 to 17, 21 to 26 or 28 to 33.

45. The kit according to any of claims 39 to 44, further characterized in that it additionally comprises third and / or fourth oligonucleotide primers as defined in claim 6 or 7.

46. ​​A device containing a kit of any of claims 39 to 10.

45.

47. The device according to claim 46, further characterized in that it is an energized device.

48. The device according to claim 46 or 47, further characterized in that it comprises heating means. 15 49. The device according to any of claims 46 to 48, further characterized in that it is a single-use diagnostic device.