nucleic acid detection method

Abstract

The present invention relates to a method for detecting nucleic acids of a predetermined sequence and to a kit and apparatus for use in said method. The present method uses a restriction enzyme, a polymerase, and an oligonucleotide primer to produce an amplification product in the presence of a target nucleic acid that is in contact with an oligonucleotide probe to produce a detection product.

Description

Technology Field

[0001] The present invention relates to a method for detecting nucleic acids of a predetermined sequence, and a kit and apparatus for use in said method. Background Technology

[0002] 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 utilizes temperature cycling to achieve primer annealing, elongation by DNA polymerase, and denaturation of newly synthesized DNA during the cycling exponential amplification process. The requirement for temperature cycling necessitates complex equipment, which limits the use of PCR-based methods in certain application areas.

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

[0004] In SDA, the 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 the restriction enzyme is used to form a nick at the restriction site due to its ability to cleave only the unmodified strand in the hemiphosphorothioate form of the recognition site. A strand replacement polymerase extends the 3' end of each nick and replaces the downstream DNA strand. Exponential amplification is the result of combining sense and antisense reactions, where the strand replaced from the sense reaction acts as the target for the antisense reaction and vice versa. Since SDA typically takes more than one hour to perform, its potential in clinical diagnostics is significantly limited. Furthermore, the requirement for separate processes for the specific detection of the post-amplification product and reaction initiation adds significant complexity to this method.

[0005] Maples et al. (WO2009 / 012246) subsequently performed SDA using nicking enzymes, a subclass of restriction enzymes capable of cleaving only one of the two DNA strands after binding to a specific double-strand recognition sequence. They referred to this method as the NEAR (Nicking and Extension Amplification Reaction). NEAR, which uses nicking enzymes instead of restriction enzymes, has also been applied by others who attempted to improve the method by using software-optimized primers (WO2014 / 164479) and through warming initiation or controlled temperature reduction (WO2018 / 002649). However, since only a very small number of nicking enzymes are available, it is more difficult to find enzymes with characteristics suitable for specific applications.

[0006] A critical disadvantage of SDA using restriction enzymes or NEARs is that it generates double-stranded nucleic acid products, which does not provide a unique process for the efficient detection of amplified signals. This has significantly limited its utility, for example, in low-cost diagnostic devices. The double-stranded nature of the generated amplified products poses a problem in combining amplification methods with signal detection, as hybridization-based detection cannot be performed without first separating the two strands. Therefore, more complex detection methods, such as molecular beacons and fluorophore / quencher probes, are required; however, this necessitates separate process steps, making analysis protocols complex and potentially significantly reducing the potential for developing multiplex analysis.

[0007] To overcome the limitations of SDA, there is an important need for an enhanced amplification method for high-speed, responsive, and specific nucleic acid sequence detection. The present invention relates to a method for amplifying and detecting a target nucleic acid sequence, which generates a detection species that enables efficient signal detection by using an additional oligonucleotide probe in addition to a pair of primers having a 5' restriction site.

[0008] A method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample of the present invention,

[0009] a) to generate an amplification product without temperature cycling in the presence of the above target nucleic acid

[0010] i. A first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid;

[0011] ii. strand replacement DNA polymerase;

[0012] iii. dNTP;

[0013] iv. One or more modified dNTPs;

[0014] v. A first restriction enzyme that is not a nick-forming enzyme, but is capable of recognizing the recognition sequence of a first primer and cleaving only the first primer strand at the cleaving site when the recognition sequence and the cleaving site are double-stranded, wherein the cleaving of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a DNA polymerase using one or more modified dNTPs; and

[0015] vi. A second restriction enzyme that is not a nick-forming enzyme, but where the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and cleave only the second primer strand at the cleavage site, wherein the cleavage of the anti-complement strand is blocked by the presence of one or more modifications incorporated into the anti-complement strand by a DNA polymerase using one or more modified dNTPs.

[0016] Step of contacting the sample;

[0017] b)

[0018] i. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and

[0019] ii. A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence among the amplification products of at least one species, and attached to a solid material or a moiety that enables attachment to a solid material.

[0020] as a step of contacting the amplification product of step a);

[0021] Hybridization of the first and second probes for at least one species in the amplification product to generate a detection species; and

[0022] c) a step of detecting the presence of a detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample, thereby providing a method.

[0023] An embodiment of the present method is illustrated in FIG. 1.

[0024] In various embodiments, in the presence of a target nucleic acid, the present method rapidly generates multiple copies of a detection species ideally suited for responsive detection.

[0025] In various aspects, the present invention is advantageous over known methods because, in addition to providing a unique process for the efficient detection of amplified products, it includes high-speed amplification without temperature cycling.

[0026] The method of the present invention overcomes the major disadvantages of SDA, including SDA by NEAR, which is that SDA does not provide an intrinsic process for efficient detection of the amplified signal due to the double-stranded nature of the amplification product. The present invention overcomes this limitation by utilizing two additional oligonucleotide probes that hybridize to at least one species within the amplification product to facilitate high-speed specific detection. The use of these two additional oligonucleotide probes, in which the first is attached to a moiety enabling detection and the second is attached to a solid material or a moiety enabling attachment to a solid material, provides a number of additional advantages of the present invention compared to known methods such as SDA. For example, in an embodiment of the present invention in which one of the oligonucleotide probes is cut off from elongation by DNA polymerase at the 3' end, is not cleaved by restriction enzyme(s), and contacts the sample simultaneously with the performance of step a), a pre-detection species containing a single-stranded region is efficiently generated without surprisingly significant and adverse amplification inhibition being observed. This aspect of the present invention is not easy to apply because it can be assumed that such blocked probes will lead to asymmetric amplification biased toward the amplification product strand opposite to that contained in the pre-detection species. In fact, since the exposed single-stranded region is readily available for hybridization with other oligonucleotide probes, the pre-detection species is efficiently generated and is ideally suitable for efficient detection.

[0027] The unique sample detection approach of the present method is fundamentally different from previous attempts to overcome the critical limitations of SDA, which include the step of performing "asymmetric" amplification using unequal primer ratios to generate, for example, an excess of one amplicon strand compared to another. The present method does not require asymmetric amplification and does not require generating an excess of one amplicon strand compared to another; instead, it focuses on the generation of detection species after hybridization of the first and second oligonucleotide probes. The unique sample detection approach of the present method, which includes the generation of detection species, provides a simple, low-cost, and high-speed means of performing detection in step c), for example, by printing the second oligonucleotide probe onto a lateral flow strip, and is ideally suited for coupling with nucleic acid lateral flow among other detection methods. When coupled with nucleic acid lateral flow, the present method also enables efficient multiplexing based on the differential hybridization of multiple second oligonucleotide probes attached to individual locations on the lateral flow strip by different sequences designed for different target nucleic acid sequences within the sample. In a further embodiment of the method, the efficiency of lateral flow detection is enhanced by using a single-stranded oligonucleotide as a moiety within a second oligonucleotide probe that enables attachment to a solid material, and by printing an inverse complement sequence for said moiety onto a strip. By the latter approach, the sequence attached to the lateral flow strip can also be defined and does not need to correspond to the sequence of the target nucleic acid(s), thereby allowing the lateral flow strip to be optimized and manufactured into a single "universal" detection system for multiple target applications. Thus, the requirement for two additional oligonucleotide probes in the method of the present invention provides many advantages over SDA, including SDA by NEAR.

[0028] Because the present invention requires the use of non-nick-forming restriction enzyme(s) and one or more modified dNTPs, it is fundamentally different from SDA performed using nick-forming enzymes (NEAR) and offers many additional advantages over such nick-forming enzyme-dependent methods. For example, a much larger number of non-nick-forming restriction enzymes are available than nick-forming enzymes, and this allows the restriction enzyme(s) to be selected from a multitude of possible enzymes to ensure they possess superior characteristics for a given application, such as reaction temperature, buffer compatibility, stability, and reaction rate (responsiveness). Due to these major advantages of the present method, the inventors were able to select restriction enzymes with lower optimal temperatures and higher rates than can be achieved with nick-forming enzymes. These restriction enzymes are much more suitable for developing low-cost diagnostic devices. Furthermore, the requirement to use one or more modified dNTPs is an essential feature of the present invention that provides significant advantages in addition to the restriction enzyme being provided to cleave only one strand of the restriction site. For example, certain modified dNTPs, such as alpha-thiol dNTPs, reduce the melting temperature (Tm) of the DNA containing them, meaning that the oligonucleotide primers and probes of the present method have a greater affinity for hybridization to species within the amplification product than any competitive complement strand containing the modified dNTP generated during amplification. Additionally, the reduction in the Tm of the amplification product due to the insertion of modified dNTP bases facilitates the separation of double-stranded DNA species, thereby improving amplification rates, reducing the optimal temperature, and enhancing responsiveness. Alternatively, other modified dNTPs may increase the Tm of the DNA into which they are incorporated, providing additional opportunities to adjust the performance of the present method for a given application.

[0029] The numerous advantages of the present invention compared to SDA using restriction enzymes or NEARs together provide utility for the present method in low-cost single-use diagnostic devices due to the simple visualization of amplified signals and improved amplification speed, which are impossible with known methods.

[0030] Various embodiments of the aforementioned aspects and additional aspects of the present invention are described in more detail below. Brief explanation of the drawing

[0031] FIG. 1. Schematic diagram of the method according to one aspect of the present invention. FIG. 2. Schematic diagram of the present method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and contacts a sample in step a). FIG. 3. Schematic diagram of steps b) and c) of the present method, in which the moiety enabling attachment of the second oligonucleotide probe and the solid material is a single-stranded oligonucleotide. FIG. 4. Schematic diagram of part of step a) of the present method in which the sample is further contacted with the third and fourth oligonucleotide primers in step a). FIG. 5. Performance of the present method in which a second oligonucleotide probe is attached to a solid material, a nitrocellulose lateral flow strip (see Example 1). FIGS. 6a and FIGS. 6b. The first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme, and the method of the present invention is performed in step a) in contact with the sample (see Example 2). FIGS. 7a, FIGS. 7b, FIGS. 7c, and FIGS. 7d. Performance of the method in which the presence of two or more different target nucleic acids of a given sequence is detected in the same sample (see Example 3). FIG. 8. Performance of the present method in which the first and second hybridization sequences in the target nucleic acid are separated by five bases (see Example 4). FIG. 9. A moiety enabling attachment of a second oligonucleotide probe to a solid material is an antigen, and the corresponding antibody is attached to a solid surface nitrocellulose lateral flow strip (see Example 5). FIG. 10a and FIG. 10b. A moiety enabling attachment of a second oligonucleotide probe to a solid material is a single-stranded oligonucleotide comprising four repeat copies of a three-base DNA sequence motif, and an inverse complement of said single-stranded oligonucleotide sequence attached to a solid material, thereby performing the method of the present invention (see Example 6). FIG. 11. Use of the present method for detection of RNA viruses in clinical specimens (see Example 7). FIG. 12a and FIG. 12b. Performance of the present method at different temperatures (see Example 8). FIGS. 13a and FIGS. 13b. Performance of the present method in which the target nucleic acid is derived from double-stranded DNA by strand penetration (see Example 9). FIG. 14a and FIG. 14b. Comparison of the performance of the method of the present invention versus a known method (see Example 10). Specific details for implementing the invention

[0032] The present invention provides a method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample. The target nucleic acid may be single-stranded DNA, comprising single-stranded DNA derived from double-stranded DNA after the dissociation of two strands in the sample, for example by thermal denaturation or through the strand replacement activity of a polymerase, or derived from RNA, for example by the action of a reverse transcriptase, or derived from double-stranded DNA by the use of a nuclease, for example, restriction endonuclease or exonuclease III, or derived from an RNA / DNA hybrid through an enzyme such as ribonuclease H. The target nucleic acid may be single-stranded DNA derived from DNA in the sample by a DNA polymerase, helicase, or recombinase. A single-stranded region within the double-stranded DNA may be sufficiently exposed for hybridization and elongation of the first oligonucleotide primer disclosing the method by, for example, "strand penetration," and sufficient transient opening of one or more DNA bases within the double-stranded DNA may be achieved to enable hybridization and elongation of the 3' hydroxyl of the first oligonucleotide primer, or to enable generative nicking of DNA or transient conversion to Hoogsteen pairs via restriction enzyme or thermochemical approaches by spontaneous opening of DNA base pairs. The target nucleic acid may be a single-stranded RNA comprising, for example, a double-stranded RNA derived by thermal denaturation after the dissociation of two strands in the sample, or a double-stranded DNA derived by, for example, transcription.

[0033] The present method comprises, in step a), (i) a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single strand of restriction enzyme recognition sequence and cleavage site and a first hybridization sequence in the 5' to 3' direction and a region capable of hybridizing to a single strand of restriction enzyme recognition sequence and cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid in the 5' to 3' direction and a region capable of hybridizing to the reverse complement of a second hybridization sequence; (ii) a strand-replacement DNA polymerase; (iii) a dNTP; and (iv) one or more modified dNTPs; (v) a first restriction enzyme that is not a nick-forming enzyme but, when the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the first primer and can cleave only the first primer strand at the cleavage site, wherein the cleavage of the reverse complement strand is blocked by the presence of one or more modifications incorporated into the reverse complement strand by DNA polymerase using one or more modified dNTPs; and (vi) a second restriction enzyme that is not a nick-forming enzyme but, when the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and can cleave only the second primer strand at the cleavage site, wherein the cleavage of the reverse complement strand is blocked by the presence of one or more modifications incorporated into the reverse complement strand by DNA polymerase using one or more modified dNTPs, and the method comprises the step of contacting a sample with the second restriction enzyme.

[0034] If the target nucleic acid detected in the sample is double-stranded, either strand may be considered as the single-stranded target nucleic acid of the present method because one of the two oligonucleotide primers can hybridize to one strand and the other oligonucleotide primer can hybridize to the other strand. Generally, the oligonucleotide primers used in the present method are DNA primers that form a hybrid dimer containing both strands of double-stranded DNA or RNA and DNA together with DNA or RNA. However, primers containing other nucleic acids, such as non-natural bases and / or alternative backbone structures, may also be used.

[0035] In the presence of the target nucleic acid, the first oligonucleotide primer hybridizes to the first hybridization sequence of the target nucleic acid. After hybridization, the 3' hydroxyl group of the first primer is extended by strand replacement DNA polymerase, or optionally by reverse transcriptase (e.g., M-MuLV) in the case of RNA target nucleic acid, to produce a double-stranded species comprising the extended first primer and the target nucleic acid (see FIG. 1). The strand replacement DNA polymerase, or reverse transcriptase if present, uses dNTPs and one or more modified dNTPs in the extension. One strand of the restriction enzyme recognition sequence and the cleavage site at the 5' end of the first primer generally do not hybridize because the corresponding reverse complement sequence is typically not present in the target nucleic acid sequence. Therefore, the first primer is generally used to introduce the one strand of the restriction enzyme recognition sequence and the cleavage site into the subsequent amplification product species. "Target removal" is performed after the extension of the first primer. Target removal allows access to the extended first primer species for hybridization of the second oligonucleotide primer and the reverse complement of the second hybridization sequence. If the target nucleic acid is RNA, target removal can be achieved, for example, by RNase H degradation of the RNA, or, if present, through the RNase H activity of the reverse transcriptase or through the separate addition of this enzyme. Alternatively, if the target nucleic acid is single-stranded DNA containing a single-stranded region within double-stranded DNA, this can be achieved by strand replacement using an additional upstream primer or bump primer.Alternatively, in particular where only short extension products are produced from a specific target nucleic acid molecule, such target removal may be achieved after spontaneous dissociation, or through strand penetration in which the transient opening of one or more DNA base pairs within the double strand is sufficiently achieved to enable hybridization and extension of the 3' hydroxyl of the second oligonucleotide primer by strand replacement.

[0036] After hybridization of the second oligonucleotide primer and the reverse complement of the second hybridization sequence, the strand replacement DNA polymerase extends the 3' hydroxyl of the primer using dNTPs and one or more modified dNTPs. The double-stranded restriction recognition sequence and cleavage site of the first restriction enzyme are formed by one or more modified dNTP base(s) incorporated into the reverse complement strand, and act to block the cleavage of the strand by the first restriction enzyme. The first restriction enzyme recognizes its recognition sequence and cleaves only the first primer strand at the cleavage site, thereby producing a 3' hydroxyl extended by the strand replacement DNA polymerase using dNTPs and one or more modified dNTPs, and replaces the first primer strand. The double-stranded restriction recognition sequence and cleavage site of the second restriction enzyme are formed by one or more modified dNTP base(s) incorporated into the reverse complement strand, and act to block the cleavage of the strand by the second restriction enzyme. Two primer sequences are positioned side by side, and a double-stranded species is thus generated in which a portion of the first restriction enzyme and the second restriction enzyme's blocked restriction sites are present. The first restriction enzyme of the first primer strand and the second restriction enzyme of the second primer strand are then cleaved, and two double-stranded species are generated, one containing the first primer sequence and the other containing the second primer sequence. Subsequent cleavage and replacement of the first primer strand and the second primer strand are then performed during the clinical amplification process, and the replaced first primer strand acts as a target for the second primer, and the replaced second primer strand acts as a target for the first primer.

[0037] In the presence of the target nucleic acid, an amplification product is generated without any requirements for temperature cycling.

[0038] An essential aspect of the present invention is that the amplification product of step a) is not directly detected, but rather the detection species is generated after specific hybridization of both the first and second oligonucleotide probes with at least one species within the amplification product. The first oligonucleotide probe attached to a moiety enabling detection hybridizes to the first single-strand detection sequence in the at least one species. The second oligonucleotide probe attached to a moiety enabling attachment to a solid material or a solid material hybridizes to the second single-strand detection sequence upstream or downstream of the first single-strand detection sequence in the at least one species.

[0039] Referring to FIG. 1, the amplification product comprises a number of different species, such as a species comprising a single-stranded detection sequence consisting of the whole or part sequence of both the first primer and the second primer or an inverse complement sequence, and it will be obvious to those skilled in the art that if the primers binding to the first and second hybridization sequences in the target nucleic acid are separated by one or more bases, the sequence may be separated by the target-derived sequence. It will also be obvious that any of the said species may be selected to hybridize to the first and second oligonucleotide probes to form the detection species.

[0040] The detection species generated in step b) is detected in step c), and the presence of the detection species indicates the presence of the target nucleic acid in the sample.

[0041] By using two oligonucleotide probes, one for detection and the other for attachment to a solid material, the method of the present invention overcomes the requirements for a more complex second detection method and provides high-speed, efficient signal detection that provides, for example, efficient visualization of a signal generated in the presence of a target by nucleic acid lateral flow.

[0042] A method of the present invention may be performed in which one of the first and second oligonucleotide probes is cut off at the 3' end from extension by strand replacement DNA polymerase and is not cleaved by the first or second restriction enzyme. Accordingly, according to further embodiments, the present invention is a method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample, wherein

[0043] a) to generate an amplification product without temperature cycling in the presence of the above target nucleic acid

[0044] i. A first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid;

[0045] ii. strand replacement DNA polymerase;

[0046] iii. dNTP;

[0047] iv. One or more modified dNTPs;

[0048] v. A first restriction enzyme that is not a nick-forming enzyme, but is capable of recognizing the recognition sequence of a first primer and cleaving only the first primer strand at the cleaving site when the recognition sequence and the cleaving site are double-stranded, wherein the cleaving of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a DNA polymerase using one or more modified dNTPs; and

[0049] vi. A second restriction enzyme that is not a nick-forming enzyme, but where the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and cleave only the second primer strand at the cleavage site, wherein the cleavage of the anti-complement strand is blocked by the presence of one or more modifications incorporated into the anti-complement strand by a DNA polymerase using one or more modified dNTPs.

[0050] Step of contacting the sample;

[0051] b)

[0052] i. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and

[0053] ii. A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence among the amplification products of at least one species, and attached to a solid material or a moiety that enables attachment to a solid material.

[0054] as a step of contacting the amplification product of step a);

[0055] One of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme, and hybridization of the first and second probes for at least one species in the amplification product generates a detection species; and

[0056] c) a step of detecting the presence of a detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample

[0057] Provides a method including

[0058] In an embodiment, the one blocked oligonucleotide probe is not cleaved by a first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate linkages. In a further embodiment, the one blocked oligonucleotide probe is in contact with the sample simultaneously with the performance of step a), that is, during the performance of step a), and is present during the generation of the amplification product in the presence of the target nucleic acid. Thus, according to a further embodiment, the present invention is a method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample,

[0059] a) to generate an amplification product without temperature cycling in the presence of the above target nucleic acid

[0060] i. A first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid;

[0061] ii. strand replacement DNA polymerase;

[0062] iii. dNTP;

[0063] iv. One or more modified dNTPs;

[0064] v. A first restriction enzyme that is not a nick-forming enzyme, but is capable of recognizing the recognition sequence of a first primer and cleaving only the first primer strand at the cleaving site when the recognition sequence and the cleaving site are double-stranded, wherein the cleaving of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a DNA polymerase using one or more modified dNTPs; and

[0065] vi. A second restriction enzyme that is not a nick-forming enzyme, but where the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and cleave only the second primer strand at the cleavage site, wherein the cleavage of the anti-complement strand is blocked by the presence of one or more modifications incorporated into the anti-complement strand by a DNA polymerase using one or more modified dNTPs.

[0066] Step of contacting the sample;

[0067] b)

[0068] i. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and

[0069] ii. A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence among the amplification products of at least one species, and attached to a solid material or a moiety that enables attachment to a solid material.

[0070] as a step of contacting the amplification product of step a);

[0071] One of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme, and is brought into contact with a sample simultaneously with the performance of step a), and hybridization of the first and second probes for at least one species in the amplification product generates a detection species; and

[0072] c) a step of detecting the presence of a detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample

[0073] Provides a method including

[0074] For example, in the embodiment illustrated in FIG. 2, the first oligonucleotide probe is blocked and hybridized to a first single-strand detection sequence of at least one species in the amplification product to form a pre-detection species comprising a single-strand region. The at least one species may be extended by a strand-replacement DNA polymerase that extends its 3' hydroxyl group, thereby further stabilizing the pre-detection species. Thus, in the embodiment, the blocked oligonucleotide probe includes an additional region that allows the 3' end of the species in the amplification product to which the blocked oligonucleotide probe hybridizes to be extended by a strand-replacement DNA polymerase. The "stabilized pre-detection species" is generated as shown in FIG. 2. Those skilled in the art will understand that in the blocked oligonucleotide probe, this additional pre-detection species stabilization region will be located upstream of the region that hybridizes to at least one species of the first or second single-strand detection sequence in the amplification product. In an embodiment using a blocked oligonucleotide probe, the hybridization sequence of the blocked oligonucleotide probe and the primers of appropriate concentrations can be optimized so that a specific proportion of related species generated in the amplification product in each cycle hybridizes to the blocked oligonucleotide probe, and the remaining copies of these species remain in a state where they can participate in the cyclic amplification process. The oligonucleotide probe is blocked from elongation, for example, by the use of a 3' phosphate modification, and in this embodiment, is also attached to a moiety that enables detection, such as a 5' biotin modification. Alternatively, a single 3' modification can be used as a moiety that blocks elongation and enables detection. Various other modifications are available to block the 3' end of the oligonucleotide, such as a C-3 spacer; alternatively, mismatched base(s) may be used.The above-mentioned pre-detection species is ideally suited for efficient detection because the exposed single-stranded region is readily available for hybridization with the second oligonucleotide probe. The second oligonucleotide probe can be attached to the nitrocellulose surface of the nucleic acid lateral flow strip, so that when the pre-detection species is introduced thereon, sequence-specific hybridization occurs easily and the detection species is positioned at a specific location on the strip. A dye attached to a detection moiety, such as carbon, gold, or polystyrene particles to which streptavidin may be present during the amplification reaction or on the conjugate pad of the nucleic acid lateral flow strip, provides high-speed color-based visualization of the presence of the detection species generated in the presence of the target nucleic acid.

[0075] In another embodiment, this is a second oligonucleotide probe that is cut off at the 3' end from extension by strand replacement DNA polymerase and is not cleaved by a first or second restriction enzyme, and is in contact with the sample simultaneously with the performance of step a). Before contacting the sample, the second oligonucleotide probe may be attached to a solid material, such as the surface of an electrochemical probe, a 96-well plate, a bead, or an array surface, or may be attached to a moiety that enables attachment to a solid material. At least one species of a specific proportion generated during amplification is hybridized to the second oligonucleotide probe after generation, instead of hybridizing to a related reaction primer to further participate in the cyclic amplification process. After hybridization to the second oligonucleotide probe, said species are extended onto the oligonucleotide probe by a polymerase to generate a stabilized pre-detection species. The first oligonucleotide probe and the detection moiety may also be in contact with the sample simultaneously with the performance of step a) and will be localized on said surface at a site of the second oligonucleotide probe. By detecting the accumulation of detection moiety at the above site during the amplification process, a real-time signal will be obtained that provides quantification of the number of copies of target nucleic acid present in the sample. Accordingly, according to an embodiment of the present invention, two or more of steps a), b), and c) are performed simultaneously.

[0076] In the execution of an embodiment in which one of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme and contacted with the sample simultaneously with the execution of step a), the inventors did not observe any significant inhibition of the amplification rate, which indicates that the pre-detection species accumulate in real time without interfering with the optimal cyclic amplification process. This differs from attempts to manipulate asymmetric SDA by utilizing unequal primer ratios to generate one amplicon strand in excess of the other. Instead of increasing the ratio of the other strand by removing one amplicon strand from the reaction using the blocked oligonucleotide probe, the present invention focuses on the generation and detection of detection species using the blocked probe, thereby facilitating the exposure of the single-strand region during the amplification process. Therefore, not only did the inventors not observe any inhibitory effect on the amplification process in the above embodiment, but the inventors also observed a remarkable improvement in which a signal corresponding to an increased amount of detection species was generated in a specific embodiment, refer to Example 2 (Fig. 6).

[0077] Additionally, the above embodiment of the method of the present invention, in which one of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme and contacts the sample simultaneously with the performance of step a), exhibits a fundamental advantage over reported attempts to incorporate NEAR by nucleic acid lateral flow in a multi-step process without blocked probes. For example, WO2014 / 164479 required a long incubation of 30 minutes at 48°C to visualize the amplification product using nucleic acid lateral flow, which appears to be a major obstacle to using this method in point-of-care diagnostic devices, particularly in low-cost or single-use devices. In contrast to this, the method of the present invention easily achieves uniform amplification in less than 5 minutes and at lower temperatures, e.g., 40 to 45°C. In an additional direct comparison study (see Example 10), the method of the present invention shows a remarkably superior rate compared to the prior art method (WO2014 / 164479) resulting from the combination of the use of a restriction enzyme other than a nick-forming enzyme, the use of modified dNTP bases, and the use of the blocked oligonucleotide probe.

[0078] Also, as described above, it will be understood that the other of the first and second oligonucleotide probes may be blocked from elongation by DNA polymerase at the 3' end and / or may not be cleaved by the first or second restriction enzyme.

[0079] An essential aspect of the present method is the use of one or more restriction enzymes that, although not a nick-forming enzyme, recognize the recognition sequence and the cleavage site when the recognition sequence and the cleavage site are double-stranded, and can cleave only one strand of the cleavage site, and the cleavage of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a strand-replacement DNA polymerase using one or more modified dNTPs that confer nuclease resistance after their incorporation by a polymerase, for example, dNTPs.

[0080] "Restriction enzymes" [or "restriction endonucleases"] are a broad class of enzymes that bind to a specific recognition sequence and cleave one or more phosphodiester bonds on one or both strands of a double-stranded nucleic acid molecule at a specific cleavage site. There are over 3,000 reported restriction enzymes and over 600 commercially available restriction enzymes available, which include various physicochemical properties and recognition sequence specificities.

[0081] "Nick-forming enzymes" [or "nick-forming endonucleases"] are specific subtypes of restriction enzymes that, after binding to a specific recognition sequence, can cleave only one strand of a double-stranded nucleic acid molecule at a specific cleavage site without cleaving the other strand. Only a very small number (c.10) of nick-forming enzymes are available, including both naturally occurring and engineered enzymes. Nick-forming enzymes include downstrand cleavages Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, and Nb.BtsI, and upstrand cleavages Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.

[0082] Although restriction enzymes other than nick-forming enzymes used exclusively in the method of the present invention can also cleave both strands of a double-stranded nucleic acid, under certain circumstances, after binding to their recognition sequence, they may cleave only one strand of their double-stranded DNA cleavage site or form a nick. This can be accomplished in a number of ways. Particularly in relation to the present method, this can be achieved by modifying one strand of a double-stranded nucleic acid target site using nuclease-resistant modifications, such as phosphothioate (PTO), boranophosphate, methylphosphate, or peptide-nucleotide linkages, so that the phosphodiester bond at the cleavage site on one strand is protected, thereby preventing one of the strands within the double-stranded nucleic acid from being cleaved at the cleavage site. Specific modified nucleotide linkages, such as PTO linkages, may be chemically synthesized within oligonucleotide probes and primers or incorporated into the double-stranded nucleic acid by a polymerase, such as through the use of one or more alpha-thiol modified deoxynucleotides. Accordingly, in the embodiments, one or more modified dNTPs are alpha-thiol modified dNTPs. Typically, the S isomer is used, which is incorporated to more effectively confer nuclease resistance.

[0083] Due to the very large number of available restriction enzymes other than nick-forming enzymes, a wide range of enzymes with different characteristics are available to screen for suitable performance characteristics, e.g., temperature characteristics, rate, buffer compatibility, polymerase cross-compatibility, recognition sequence, thermal stability, manufacturability, etc., for use in the present method for a given application. In contrast, the fact that only a small number of nick-forming enzymes are available limits the potential of conventional methods using nick-forming enzymes and may lead, for example, to lower reaction rates (responsiveness, time to result) and higher reaction temperatures. The restriction enzymes other than nick-forming enzymes selected for use in the present method may be naturally occurring or engineered enzymes.

[0084] In selecting a restriction enzyme other than a nick-forming enzyme for use in the present method, those skilled in the art will recognize that it is necessary to identify an enzyme having an appropriate cleavage site to ensure that the modification is included at the correct location to block the cleavage of the relevant strand rather than the other strand. For example, in embodiments where modified dNTPs, such as alpha-thiol dNTPs, are used, it may be desirable to select a restriction enzyme having a cleavage site outside the recognition sequence, such as an asymmetric restriction enzyme having a non-palindromic recognition sequence, to provide sufficient flexibility to position primers to include the modified nucleotide base at the appropriate location to block the cleavage of the relevant strand after the target nucleic acid sequence is incorporated. For example, when alpha-thiol dATPs are used, in order to ensure that the primer is properly cleaved during the performance of the present method, the reverse complement sequence of the restriction enzyme cleavage site in the relevant oligonucleotide primer includes an adenosine base downstream of the cleavage site in the reverse complement strand, but does not include an adenosine base downstream of the cleavage site in the primer sequence. Accordingly, asymmetric restriction enzymes having a non-palindromic recognition sequence that is cleaved outside the recognition sequence are ideally suitable for use in the present invention. A partial or degenerate palindromic sequence that recognizes a restriction enzyme cleaved within the recognition site may also be used. Nuclease-resistant nucleotide linkage modifications, e.g., PTO, may be used to block the cleavage of either strand by a wide range of commercially available double-strand cleavage agents of various different classes, including IIS-type and IIG-type restriction enzymes having both partial or degenerate palindromic and asymmetric restriction recognition sequences, in order to enable their use in the method of the present invention.

[0085] Restriction enzyme(s) are generally used in the present method in amounts ranging from 0.1 to 100 units, where 1 unit is defined as the amount of agent required to degrade 1 μg T7 DNA within 1 hour at a given temperature (e.g., 37°C) in a total reaction volume of 50 μl. However, the above amounts depend on several factors such as the activity of the selected enzyme, the concentration and type of the enzyme, the expected target nucleic acid concentration, the reaction volume, the primer concentration, and the reaction temperature, and should not be considered as limiting in any way. Those skilled in the art will understand that the restriction enzyme used in the present method requires suitable buffers and salts, e.g., divalent metal ions, effective and efficient function, pH control, and enzyme stabilization.

[0086] In the embodiments, the first and second restriction enzymes are the same restriction enzyme. By using only a single restriction enzyme, the present method is simplified in many ways. For example, it is sufficient to identify only a single enzyme compatible with other reaction components, optimize it for the execution of the present method, and manufacture and stabilize it. Furthermore, using a single restriction enzyme simplifies the design of oligonucleotide primers and supports symmetry in the amplification process.

[0087] In this method, the restriction enzyme cleaves only one strand of the nucleic acid dimer, thereby providing an exposed 3' hydroxyl group that can serve as an efficient priming site for the polymerase after cleavage. The polymerase is an enzyme that synthesizes a chain or polymer of nucleic acids by extending the primer and using base pair interactions to generate an anti-complement "copy" of the DNA or RNA template strand. Polymerases with strand replacement ability are used in the performance of this method to appropriately replace strands to influence the amplification process. The term "strand replacement" refers to the polymerase's ability to replace downstream DNA encountered during synthesis. Various polymerases with strand replacement ability operating at different temperatures have been characterized and are commercially available. For example, the Phi29 polymerase has very strong strand replacement ability. Polymerases of Bacillus species, such as Bst DNA Polymerase Large Fragment, generally exhibit high strand replacement activity and are suitable for use in the performance of this method. The E. coli (E. coli) Klenow fragment (exo-) is another widely used strand replacement polymerase. For example, strand replacement polymerases such as KlenTaq can be easily engineered by cloning only the relevant active polymerase domain of the endogenous enzyme and knocking out any exonuclease activity. For the execution of the present method where the single-stranded target nucleic acid is RNA, RNA-dependent DNA synthesis (reverse transcriptase) activity is also required, and this activity can be performed by the strand replacement polymerase and / or a separate additional reverse transcriptase, e.g., M-MuLV or AMV, in step a).

[0088] Polymerase(s) are generally used in the relevant steps of this method in appropriate amounts that are optimized according to the enzyme, reagent concentrations, and appropriate reaction temperature. For example, 0.1 to 100 units of Bacillus polymerase may be used, where 1 unit is defined as the amount of enzyme capable of incorporating 25 nmol of dNTPs into an acid-insoluble substance within 30 minutes at 65°C. However, the above amounts depend on various factors such as the activity, concentration, and type of the polymerase, the expected target nucleic acid concentration, the reaction volume, the number and concentration of oligonucleotide primers, and the reaction temperature, and should not be considered as limiting in any way.

[0089] Those skilled in the art will know that dNTP monomers are required for the polymerase to possess polymerase activity, and that an appropriate buffer solution is also required along with components such as buffer salts, divalent ions, and stabilizers. Additionally, one or more modified dNTPs are used in this method to block the cleavage of the primer's anti-complement strand after incorporation by strand-replacement polymerase. Generally, when a single modified dNTP is used, the dNTP used in this method must be missing the corresponding base. For example, in an embodiment where the modified dNTP is alpha-thiol dATP, the dNTP must contain only dTTP, dCTP, and dGTP and not dATP. Removal of the corresponding natural dNTP base blocks all necessary downstrand cleavage sites within the primer's anti-complement sequence, because only the modified base is available for incorporation by the polymerase, but the complete or partial removal of the corresponding natural dNTP is not necessarily achieved. dNTPs may generally be used in this method at concentrations similar to those used in other polymerase methods, such as concentrations in the range of 10 micromolar to 1 millimolear; however, the concentration of dNTPs for this method may be optimized for any given enzyme and reagent to maximize activity and minimize initial synthesis to prevent background signal generation. Given that a specific polymerase may exhibit a lower incorporation rate with one or more modified dNTP bases, one or more modified bases may be used in this method at higher relative concentrations than unmodified dNTPs, e.g., five times higher, but this should be considered non-limiting.

[0090] The use of one or more modified dNTPs is an essential feature of the present invention, providing significant advantages in addition to the restriction enzyme being provided to cleave only one strand of the restriction site. For example, certain modified dNTPs, such as alpha-thiol dNTPs, reduce the melting temperature (Tm) of the DNA into which they are incorporated, which means that the oligonucleotide primers and probes used in the present method have a greater affinity for hybridization to species within the amplification product than any competitive modified dNTP complement strand generated during amplification. For example, this key feature improves the amplification rate because, when one of the substituted strands hybridizes to the reverse complement to generate a "non-genetic" endpoint species, it dissociates more easily than the "genetic" hybridization of said substituted strand to an additional primer due to the presence of one or more modified bases, thereby reducing the Tm of the hybridization. It has been reported that phosphothioate internucleotide linkages can cause significant changes in physicochemical properties by reducing Tm, the temperature at which exactly half of the single strand of a duplex hybridizes, by 1 to 3°C per addition. The inventors have also observed an enhanced strand replacement rate when phosphothioate nucleotide linkages are present in the DNA sequence. Furthermore, the oligonucleotide probes used in the present method possess a higher affinity for the corresponding species within the amplification product than any competing modified species, regardless of whether they come into contact with the sample simultaneously with or subsequently with the performance of step a), and thus can facilitate the generation of the detected species by preferentially hybridizing or even replacing the hybridized strand. The enhanced replacement of the amplification product species and the reduction in Tm resulting from the modified internucleotide linkages included in them serve to fundamentally improve the speed of the present method and reduce the temperature required for high-speed amplification.

[0091] In addition to the speed enhancement resulting from the use of one or more modified nucleotides, the hybridization specificity of the oligonucleotide primers and probes of the present method is also enhanced. Given that all bases of a specific nucleotide are generally substituted within the amplification product, the hybridization sites of the primers and probes typically contain modified bases, and the reduced Tm resulting from the linkage between phosphorothioate nucleotides means that, for example, sequence mismatches caused by non-specific hybridization are rarely allowed.

[0092] Accordingly, the essential feature of the method of the present invention for one or more modified dNTPs is that it improves the responsiveness and specificity of amplification and is completely different from known methods without the need for modified nucleotides, such as NEAR (WO2009 / 012246), including NEAR variants (WO2014 / 164479) or heating initiation or controlled temperature reduction (WO2018 / 002649) by software-optimized primers.

[0093] There are a number of different modified dNTPs, such as modified dNTPs that confer nuclease resistance after polymerase incorporation, and they can be used in the method to achieve resistance to restriction enzyme cleavage and other properties that improve the performance of the method for a given application in the embodiments. In addition to alpha-thiol dNTPs that provide nuclease resistance and Tm reduction, modified dNTPs that are polymerase-incorporable and reported to confer nuclease resistance include equivalent nucleotide derivatives such as borano derivatives, 2'-O-methyl (2'OMe) modified bases, and 2'-fluoro bases. Other modified dNTPs or equivalent compounds that may be incorporated by a polymerase and used in embodiments of the method to enhance specific properties of the method include those that decrease binding affinity, e.g., inosine-5'-triphosphate or 2'-deoxyzebularin-5'-triphosphate; those that increase binding specificity, e.g., 5-methyl-2'-deoxycytidine-5'-triphosphate or 5-[(3-indolyl)propionamide-N-allyl]-2'-deoxyuridine-5'-triphosphate; and those that enhance the synthesis of GC-rich regions, e.g., 7-deaza-dGTP. Specific modifications may increase Tm, which may provide additional possibilities for controlling hybridization events in embodiments of the method.

[0094] Steps a), b), and c) can be performed over a wide range of temperatures. The optimal temperature for each step is determined by the optimal temperatures of the relevant polymerase and restriction enzyme and the melting temperature of the hybridization region of the oligonucleotide primer. In particular, the present method does not use temperature cycling in step a). Furthermore, the amplification step a) does not require any controlled temperature change, any high temperature or heating initiation, preheating, or controlled temperature reduction. Using the present method, steps can be performed over a wide temperature range, e.g., from 15°C to 60°C, e.g., from 20°C to 60°C, or from 15°C to 45°C. According to one embodiment, step a) is performed at a temperature of 50°C or lower, or about 50°C. Considering the various restriction enzymes other than nick-forming enzymes available in the present method, restriction enzymes that are faster at relatively lower temperatures compared to alternative methods using nick-forming enzymes can be selected. Using one or more modified nucleotides also reduces the required amplification temperature. In addition to having the potential for lower optimal temperature characteristics compared to known methods, the method of the present invention can be performed over an unusually wide temperature range. Since controlled heating imposes complex physical constraints that increase the product cost of such devices to a point where single-use or device-free devices are commercially unfeasible, these characteristics make the use of the present method in low-cost diagnostic devices very attractive. For example, many analyses have been developed using a method capable of performing high-speed detection of target nucleic acids at ambient temperature or about 37°C. As such, in additional embodiments, step a) is performed at a temperature of 45°C or lower or about 45°C. To simplify user steps and reduce the total time to result, it may be desirable to initiate the method at a temperature lower than the target temperature. As such in additional embodiments of the present method, the temperature of step a) is increased during amplification.For example, the temperature of the method may start at an ambient temperature such as 20°C and increase over a period of time, for example, 2 minutes, to a final temperature such as about 45°C or 50°C. In an embodiment, the temperature is increased during the performance of step a), for example, from an ambient starting temperature to a temperature in the range of, for example, 15 to 30°C, up to a maximum of 40 to 50°C.

[0095] The low-temperature capability and versatility of the method of the present invention, unlike known methods, mean that it may be compatible with the conditions required for various other analyses, such as immunoassays or enzymatic analyses for the detection of other biomarkers, such as proteins or small molecules. Thus, the present method can be used, for example, for the simultaneous detection of target small molecules or both nucleic acids and proteins within a sample. Components required to perform the present method, including non-knock-forming restriction enzymes, strand-replacement DNA polymerases, oligonucleotide primers, oligonucleotide probes, dNTPs, and one or more modified dNTPs, may be freeze-dried or lyophilized for stable storage and then re-hydrated to trigger the reaction, such as upon the addition of the sample. Such freeze-drying or lyophilization for stable storage generally requires the addition of one or more excipients, such as trehalose, before drying the components. A very wide range of such excipients and stabilizers for freeze-drying or lyophilization are known and may be tested to determine a composition suitable for the components required to perform the present method.

[0096] It will be obvious to those skilled in the art that the method of the present invention, which is a polymerase-based amplification method, can be improved by adding one or more additives that have been shown to improve PCR or other polymerase-based amplification methods. These additives include, but are not limited to, tetrahydrothiophene 1-oxide, L-lysine free base, L-arginine, glycine, histidine, 5-aminovaleric acid, 1,5-diamino-2-methylpentane, N,N'-diisopropylethylenediamine (TEMED), tetramethylammonium chloride, tetramethylammonium oxylate, methylsulfoneacetamide, hexadecyltrimethylammonium bromide, betaine aldehyde, tetraethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, tetrabutylammonium chloride, tetrapropylammonium chloride, formamide, dimethylformamide (DMF), N-methylformamide, N-methylacetamide, N,N-dimethylacetamide, L-threonine, N,N-dimethylethylenediamine, 2-pyrrolidone, HEP It includes (N-hydroxyethylpyrrolidone), NMP (N-methylpyrrolidone) and 1-methyl, 1-cyclohexyl-2-pyrrolidone (pyrrolidinone), δ-valerolactam, N-methylsuccinimide, 1-formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose, glycerol, Tween-20, DMSO, betaine, and BSA.

[0097] The inventors' research has revealed that this method is effective across a wide range of target nucleic acid levels, including detection down to very low, even single-copy numbers. Oligonucleotide primers are typically provided in a very high excess for the target nucleic acid. Generally, the concentration of each primer should be considered non-limiting, but is in the range of 10 to 200 nM. Higher primer concentrations can improve hybridization efficiency and thus increase the reaction rate. However, since non-specific background effects, such as primer dimers, can also be observed at high concentrations, the concentrations of the first and second oligonucleotide primers form part of the optimization process for any given analysis using this 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. Although the reaction rate may be reduced in embodiments where one primer is provided in excess of the other due to the natural symmetry of the periodic amplification process, in certain situations this may be used to reduce non-specific background signals in the method and / or to enhance the ability of the first and second oligonucleotide probes to hybridize to generate a detection species. It is desirable that the two primers be present at an unrestricted level before generating a sufficient detection species to be detected by the selected detection means.

[0098] There are many considerations regarding the design of oligonucleotide primers for performing the present method. Each first and second oligonucleotide primer must include a single strand of restriction enzyme recognition sequence, a cleavage site, and a hybridization region in the 5' to 3' direction, wherein the hybridization region can hybridize to a first hybridization region within the target nucleic acid in the case of the first primer, and to the inverse complement of a second hybridization sequence upstream of the first hybridization sequence within the target nucleic acid in the case of the second primer. Thus, a pair of primers is designed to amplify a region of the target nucleic acid. Since the restriction enzyme recognition sequence of the primer is generally not present within the target nucleic acid sequence, it forms an overhang during the initial hybridization event before being introduced into the amplicon (see Fig. 1). When an asymmetric restriction enzyme is used, the cleavage site is generally downstream of the recognition sequence and can therefore optionally be present within the hybridization sequence of the primer.

[0099] The oligonucleotide primer is designed to form an upstream primer having a melting temperature (Tm) sufficient to maintain the 5' sequence of the cleavage site hybridized to the reverse complement strand under appropriate reaction conditions after cleavage in the present method, and to replace the downstream strand of the cleavage site by the extension of the 3' hydroxyl group by the strand replacement DNA polymerase. Thus, an additional "stabilization" region may be included at the 5' end of the oligonucleotide primer, the optimal length of which is determined by the position of the cleavage site relative to the recognition sequence for the relevant restriction enzyme and other factors, such as the temperature to be used for amplification in step a). Thus, in the embodiment, the first and / or second oligonucleotide primer includes a stabilization sequence of, for example, 5 or 6 base lengths upstream of the restriction enzyme recognition sequence and the cleavage site, for example at the 5' end.

[0100] During primer design, it is necessary to ensure specific and responsive amplification in this method by defining the sequence and length of each hybridization region to enable optimal sequence-specific hybridization and strand replacement. For example, the location of the primer within the target nucleic acid to be detected in the genome of a viral or bacterial pathogen may differ in order to define the sequence of the primer's hybridization region and thus select a primer with optimal responsiveness and specificity for compatibility with the oligonucleotide probe and for amplification. Therefore, different primer pairs can be screened to identify the optimal sequence and location for performing this method. Typically, the length of the primer's hybridization region is designed so that the theoretical Tm enables efficient hybridization at an appropriate reaction temperature while allowing for easy replacement after cleavage. During primer design, the theoretical Tm of the hybridization sequence and the sequence of the replaced strand are considered in relation to the possible reaction temperature and the selected restriction enzyme, balancing theoretical improvements in sequence-derived binding specificity, which may occur as the sequence length increases. Various studies by the inventors have demonstrated significant variability in the design of primers to be effectively used in this method. In the embodiments, the hybridization region of the first and / or second oligonucleotide primer is 6 to 30, e.g., 9 to 16 bases long. In additional embodiments, modifications such as non-natural bases and alternative nucleotide linkages or base-free sites may be used in the hybridization region of the primer to improve the functionality and properties of the method for specific applications. For example, modifications that enhance Tm, such as PNA, LNA, or G-clamp, may enable shorter amplicons and thus enable shorter and more specific primer hybridization regions that improve amplification rates.

[0101] Various studies by the inventors have revealed that the speed and responsiveness of the present method can be improved by having a short amplicon, and thus it may be desirable to shorten the overall length of the primer, including its hybridization sequence in certain embodiments, and to place a primer having only a short gap, such as 10 or 15 nucleotide bases or fewer, between the first and second hybridization sequences in the target nucleic acid. In an embodiment, the first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 or 0 to 6 bases, and in a certain embodiment, they are separated by 3 to 15 or 3 to 6 bases, for example, 5, 7, or 11 bases. In further embodiments, the hybridization sequences overlap, for example, by 1 to 2 bases.

[0102] There are many considerations regarding the sequence design of the oligonucleotide probe for use in the present method. First, the region of the first oligonucleotide probe hybridizing to the first single-strand detection sequence and the region of the second oligonucleotide probe hybridizing to the second single-strand detection sequence are generally designed so that they do not overlap or overlap minimally, allowing the two oligonucleotide probes to bind simultaneously to at least one species within the amplification product. They are also generally designed to hybridize primarily to the sequence existing between the location of the cleavage site on one strand of the amplification product species and the location opposite the cleavage site on the reverse complement strand, thereby ensuring that one or more species within the amplification product are efficiently targeted, and so that the two oligonucleotide probes bind to the same strand. For any given primer pair, either strand may be selected for targeting by the oligonucleotide probe. Given that the oligonucleotide probe is generally not extended by a polymerase in the present method, the hybridization sequence is designed based on the sequence of the relevant species within the amplification product to determine Tm, %GC, and the acquired experimental data. In an embodiment, the hybridization sequences of the first and second oligonucleotide probes are 9 to 20 nucleotide bases long. In an embodiment where the first and second hybridization sequences of the target nucleic acid are separated by 0 bases, the sequence of the hybridization region of one of the oligonucleotide probes corresponds to one of the oligonucleotide primers, and the hybridization region of the other oligonucleotide probe may correspond to the anti-complement of the other oligonucleotide primer. However, the length of the hybridization sequence may be truncated to optimize the characteristics of the oligonucleotide probes for a suitable embodiment of the method and to prevent any inhibitory effect when all or part of step b) is performed simultaneously with step a).If the first or second oligonucleotide probe comprises a recognition sequence and a cleavage site for the first or second restriction enzyme and the oligonucleotide probe is brought into contact with a sample simultaneously with the performance of step a), the cleavage site within the probe is generally blocked, for example, during the introduction of a mismatch to remove the recognition sequence or during the chemical synthesis of the probe, by the inclusion of modified nucleotide-nucleotide linkages, for example, phosphorothioate linkages. Aside from hybridization regions, there is considerable variability regarding the sequences of the oligonucleotide probes and any modified nucleotide bases, nucleotide linkages, or other modifications that they may include. Modified bases that can be chemically inserted into the oligonucleotide to alter properties, such as 2-amino-dA, 5-methyl-dC, Super T®, 2-fluoro bases, and G clamps, and which can be used in embodiments of the present method, provide an increase in Tm, while others, such as iso-dC and iso-G, can improve binding specificity without increasing Tm. Other modifications, such as inosine or base-free sites, may reduce the specificity of binding. Modifications known to confer nuclease resistance include inverted dT and ddT and C3 spacers. Modifications may increase or decrease Tm and may provide the possibility for controlling hybridization events in embodiments of the method. The use of modified bases within the hybridization region of an oligonucleotide probe provides an opportunity to improve the performance of the oligonucleotide probe, for example, by enhancing binding affinity without increasing the length of the hybridization region. In one embodiment, modified bases within one or both of the oligonucleotide probes enable them to hybridize and consequently compete more effectively than any species in the amplification product that is complementary to the relevant single-stranded detection sequence.

[0103] In an embodiment in which one of the first and second oligonucleotide probes is cut off from elongation at the 3' end so as not to be cleaved and contacts the sample simultaneously with the performance of step a), typically the one oligonucleotide probe will include an additional 5' region that provides an opportunity for stabilization of the pre-detection species as described (see FIG. 2). In an embodiment, the one oligonucleotide probe includes the exact sequence of one of the oligonucleotide primers, but includes a modification that blocks elongation by strand-replacement DNA polymerase at the 3' end and a single phosphorothioate internucleotide linkage that blocks restriction enzyme cleavage sites. This embodiment simplifies the analysis design and ensures that additional sequence motifs that could lead to non-specific background amplification are not introduced.

[0104] The first and second oligonucleotide probes generating the detection species are preferably provided at a level such that the number of copies of the generated detection species sufficiently exceeds the detection limit of the means used so that the detection species can be easily detected. Furthermore, the efficiency of hybridization by the first and / or second oligonucleotide probe(s) is influenced by their concentration. Typically, the concentration of the oligonucleotide probe in contact with the sample simultaneously with the performance of step a) may be similar to the concentration of the oligonucleotide primer, e.g., 10 to 200 nM, but should be considered as having no limit. In an embodiment, the concentration of one or two oligonucleotide probes is provided in excess of the concentration of one or two oligonucleotide primers, and in another embodiment, the concentration of one or two oligonucleotide probes is provided at a lower concentration than the concentration of one or two oligonucleotide primers. If one or two oligonucleotide probes come into contact with the sample after the performance of amplification step a), higher concentrations may be enabled as needed to achieve the most efficient hybridization without considering that any inhibition of amplification step a) may result.

[0105] Hybridization sequences are a key characteristic of both oligonucleotide primers and oligonucleotide probes for performing the present method. 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 through hydrogen bond base pairs between complementary bases in the sequence of each nucleic acid. Common base pairs are adenine-thymine (AT), or adenine-uracil in the case of RNA or RNA / DNA hybrid dimers, and cytosine-guanine (CG), but a wide range of natural and non-natural analogs of nucleic acid bases having specific binding affinities are known. Furthermore, in the present invention, the complementary region of an oligonucleotide probe or primer does not necessarily have to contain a completely natural nucleic acid base in a sequence having complete and accurate complementarity to its hybridization sequence in the target nucleic acid or species within the amplification product; Rather, for the performance of the present method, the oligonucleotide probe / primer must be capable of sequence-specific hybridization with the target hybridization sequence sufficient to form the double-stranded sequence required for the precise function of the present method, including cleavage by restriction enzymes and extension by strand-replacement DNA polymerases. Thus, such hybridization may be possible using non-natural base or base-free regions without exact complementarity. In embodiments, the hybridization region of the oligonucleotide primer or oligonucleotide probe used in the present 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, where appropriate, its inverse complementary sequence. In other embodiments, one or more non-complementary base pairs are present. In some situations, it may be advantageous to use a mixture of oligonucleotide primers and / or probes in the present method.Therefore, for example, in the case of a target nucleic acid containing a single nucleotide polymorphism (SNP) site having two polymorphic positions, a 1:1 mixture of different oligonucleotide primers and oligonucleotide probes at those positions (each component is complementary to each base of the SNP) can be used. It is a common practice to randomize one or more bases during the synthesis process while preparing oligonucleotides.

[0106] Those skilled in the art will understand that amplification processes involving polymerases may include non-specific background amplification, such as that resulting from initial synthesis and / or primer-primer binding. The method of the present invention typically shows that amplification is faster when the length of the amplicon is designed to be as short as possible, for example, by minimizing the hybridization sequence of the primer, the gap between the first and second hybridization sequences in the target nucleic acid, and the length of any stabilization region while still maintaining function at a given reaction temperature. Using a shorter amplicon may exacerbate non-specific background due to the fact that all sequences necessary to generate the amplified product species are provided by the oligonucleotide primer. False positive results may occur in the present method if the amplicon is generated in a non-target-specific manner, including both the first oligonucleotide primer and the second oligonucleotide primer that are "linked" via initial synthetic DNA or primer-primer binding. The use of two oligonucleotide probes in the present method enables various embodiments of the present method that include additional characteristics to minimize any possibility of non-target-specific background signals. This embodiment, made possible by using two oligonucleotide probes, provides substantial advantages over methods known in this regard.

[0107] One approach is to separate the first and second hybridization sequences within the target nucleic acid and provide target-based sequence specificity confirmation using the oligonucleotide probe of the present method. Thus, in the embodiments, the first and second hybridization sequences within the target nucleic acid are separated by 3 to 15 or 3 to 6 bases, e.g., 5, 7, or 11 bases. This gap between the primers provides additional specificity confirmation for the species within the amplification product and provides an optimal size gap that maintains the enhanced rate of the short amplicon. Thus, in the embodiments, the first or second single-strand detection sequence of at least one species within the amplification product in step b) comprises at least 3 bases of the sequence corresponding to the 3 to 15 or 3 to 6 bases. For example, the inventors have demonstrated the possibility of distinguishing between a specific target-dependent amplification product and a non-target-specific background amplification product, as presented in Example 4 (Fig. 8).

[0108] In an alternative approach, the concentration of the first and / or second oligonucleotide primers is reduced to decrease the background probability resulting from initial amplification and primer-primer binding. To maintain the amplification rate, an additional oligonucleotide primer that is blocked at the 3' end from elongation by strand-replacement DNA polymerase may be used. In this embodiment, the unblocked first and second oligonucleotide primers are available at a sufficient concentration for the initial hybridization and elongation events to generate an amplicon from the target nucleic acid, but subsequent amplification is preferably carried out with the blocked primers provided at a higher concentration, and the cleavage of the blocked primers is performed before elongation and strand replacement to remove the 3' blocking modification and allow the amplification process to proceed without damage (see FIG. 4). Accordingly, in an embodiment, the sample is further contacted in step a) (A) a third oligonucleotide primer comprising a recognition sequence and a cleavage site of one strand of a first restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, said third oligonucleotide primer which is blocked from extension by DNA polymerase at the 3' end; and / or (B) a fourth oligonucleotide primer comprising a recognition sequence and a cleavage site of one strand of a second restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to the reverse complement of a second hybridization sequence in the target nucleic acid, said fourth oligonucleotide primer which is blocked from extension by DNA polymerase at the 3' end. In additional embodiments, if provided, the third oligonucleotide primer is provided in excess of the first oligonucleotide primer, and if provided, the fourth oligonucleotide primer is provided in excess of the second oligonucleotide primer.By substantially reducing the concentration of the first and second oligonucleotide primers, supplemented by the presence of the third and fourth oligonucleotide primers, the maximum possible benefit is obtained in relation to the elimination of non-target-dependent background amplification. For example, in addition to the presence of a 3' modification to block polymerase elongation, which can be easily achieved through the use of 3' phosphate or C-3 modifications during oligonucleotide primer synthesis, the same design parameters used for the first and second primers are applied to the third and fourth primers.

[0109] As described above, embodiments of the method of the present invention, which provide enhanced specificity and the elimination of background amplification, provide improved sequence verification accuracy, enabling low-temperature reactions to be performed without loss of specificity and / or enabling increased multiplexing when multiple reactions are performed for the simultaneous detection of multiple targets. This advantage of accurate specificity also implies that the present method may allow for a wide temperature range and suboptimal conditions (e.g., reagent concentration) without loss of specificity. For example, the inventors performed a method of increasing or decreasing the concentration of all components by 20%, and in each case, performed the present method for a significant time at ambient temperature after performing amplification in step a). Thus, these embodiments demonstrate a significant advantage of the method of the present invention over known methods, which means it is ideally suitable for use in low-cost and / or single-use diagnostic devices.

[0110] In step c), the detection of the detection species may be achieved by any technique that differentially detects the presence of the detection species from other reagents and components present in the sample. Alternatively, the presence or level of the detection species may be inferred from the depletion of one or more reaction components, such as the first or second oligonucleotide probe. Among the wide range of physicochemical techniques available for the detection of the detection species, techniques capable of generating a responsive signal present only after hybridization of the first oligonucleotide probe and the second oligonucleotide probe for the relevant species within the amplification product are preferred for use in the present method. It will be apparent to those skilled in the art that various colorimetric or fluorescent dyes exist that can be easily attached to the first oligonucleotide probe and form the basis of detection visually or using instruments such as absorbance or fluorescence spectrometers.

[0111] Accordingly, in the embodiment, the moiety enabling the detection of the first oligonucleotide probe is a moiety capable of attaching to a colorimetric or fluorescent dye or a colorimetric or fluorescent dye such as biotin.

[0112] Embodiments of the present method using a colorimetric dye have the advantage of not requiring instruments to perform fluorescence excitation and detection, and possibly allowing the presence of the target nucleic acid to be measured visually. Colorimetric detection can be achieved by directly attaching a colorimetric dye or moiety capable of attaching to the colorimetric dye before using the first oligonucleotide probe in the present method, or alternatively by specifically attaching or binding a dye or moiety to the probe fragment after cleavage. For example, the first oligonucleotide probe may include a biotin moiety that enables binding to a streptavidin-conjugated colorimetric dye for subsequent detection. One example of a colorimetric dye that can be used for detection is gold nanoparticles. A similar method can be used with various other intrinsic colorimetric moietys, of which a very large number are known, such as carbon nanoparticles, silver nanoparticles, iron oxide nanoparticles, polystyrene beads, quantum dots, etc. High extinction coefficient dyes also provide the possibility for responsive real-time quantification in the present method.

[0113] Many considerations apply when selecting a dye suitable for a given application. For example, in embodiments for performing visual colorimetric detection in solution, it is generally advantageous to select larger particles and / or particles with higher absorption coefficients for ease of detection, and embodiments including a lateral flow membrane for visual detection may benefit from the ability of smaller particles to diffuse more rapidly along the membrane. While gold nanoparticles of various sizes and shapes may be used, a number of other target colorimetric moietyes, including polystyrene or latex-based microspheres / nanoparticles, are also available. Particles of these characteristics are also available in multiple colors, which may be useful for tagging different detection species and detecting them differentially or "multiplexing" the colorimetric signals generated in the detection reaction while performing the method.

[0114] Fluorescence detection can be achieved using any dye that emits a fluorescent signal under appropriate excitation stimulation, thereby enabling subsequent detection of the detected species. 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 present method using a fluorescent dye moiety attached to an oligonucleotide probe, it is also possible to perform detection based on fluorescence resonance energy transfer (FRET), such as that used in Taqman quantitative PCR or molecular beacon-based strategies for nucleic acid detection, where the signal increases or decreases after the attachment of the detected species and the dye. Generally, when a fluorescence approach is used, the generation of the fluorescent signal can be recorded using various other detection devices, such as CCD cameras, fluorescence scanners, fluorescence-based microplate readers, or fluorescence microscopes.

[0115] In further embodiments, the moiety enabling detection of the first oligonucleotide probe is an enzyme that generates a detectable signal, such as a colorimetric or fluorescent signal, after contact with a substrate. It will be apparent to those skilled in the art that numerous enzyme-substrate systems are available and commonly used in diagnostic fields such as ELISA and immunohistochemical detection. Horseradish peroxidase (HRP) is one example. Using the enzyme attached to the first oligonucleotide probe for the detection of the detection species in step c) provides many possible advantages, such as improved detection responsiveness and increased control over signal generation through a separate step including substrate addition. Other suitable colorimetric enzymes may include glycosyl hydrolases, peptidases or amylases, esterases (e.g., carboxyesterases), glycosidases (e.g., galactosidases), and phosphatases (e.g., alkaline phosphatases). This list should not be considered limiting in any way.

[0116] In another approach, the presence of the detection species in step c) is electrically detected in the presence of the detection species, for example, by a change in impedance or a change in a signal of conductivity, current, voltage, or potential difference. Thus, in one embodiment, the detection species is detected by a change in an electrical signal. The change in the electrical signal may be facilitated by a moiety that enables the detection of the first oligonucleotide probe, such as a chemical group that induces an enhanced change in the electrical signal. Since the detection of the electrical signal may be highly sensitive, the detection moiety may simply be an oligonucleotide sequence, but in certain embodiments, the signal is enhanced by the presence of a chemical group known to enhance electrical signals, such as a metal, for example, gold and carbon.

[0117] In one embodiment, the change in the electrical signal due to the accumulation of the detection species can be detected in the aqueous reaction during amplification, but in another embodiment, the detection of the electrical signal is facilitated by localizing the detection species to a specific site, such as the surface of an electrochemical probe for detection, and said localization is mediated by a second oligonucleotide probe.

[0118] Other techniques commonly used for the detection of nucleic acids such as the detection species and which may also be used for detection in the present method include: mass spectrometry (e.g., MALDI or LC-TOF), emission spectroscopy or spectrometry, fluorescence spectroscopy or spectrometry, liquid chromatography, and fluorescence polarization.

[0119] In an embodiment, step c) uses carbon or gold, preferably carbon, to generate a colorimetric or electrochemical signal.

[0120] In the embodiments, the detection species is detected by nucleic acid lateral flow. Nucleic acid lateral flow, in which nucleic acid is separated from other reaction components by diffusion through a membrane generally made of nitrocellulose, is a low-cost, high-speed detection method that can be combined with various signal results, including colorimetric, fluorescence, and electrical signals. Nucleic acid lateral flow is highly suitable for use in the detection of the detection species in the present method and offers many advantages. In the embodiments, nucleic acid lateral flow detection is performed by attaching a colorimetric or fluorescent dye using a first oligonucleotide probe within the detection species and localizing said dye to a specific location on a lateral flow strip using a second oligonucleotide probe within the detection species. In this way, high-speed detection can be performed by observing the results visually or with a reader. For nucleic acid lateral flow, an antigen may be used as a detection moiety in the second oligonucleotide probe along with an associated antibody immobilized on the lateral flow strip. Alternatively, the present method provides a simple and low-cost alternative to antibody-based assays with improved multiplexing potential, allowing sequence-specific detection to be easily performed through hybridization of the pre-detection species or detection species into a lateral flow strip. Known methods, such as SDA, which do not use the two oligonucleotide probes of the present method, typically produce double-stranded DNA products that cannot be used for detection based on sequence-specific hybridization. Unlike the present method, multiple detection of the detection species is possible, particularly due to the use of position-specific hybridization-based detection. Carbon or gold nanoparticles can be readily used in nucleic acid lateral flow. Localization of the detection species causes localized enrichment of carbon or gold, resulting in black or red, respectively. In an embodiment, the first oligonucleotide probe comprises a moiety, such as biotin, that enables binding to a colorimetric dye before being localized on the strip by sequence-specific hybridization.

[0121] The spatial location of the detection species is closely related to the technology used for the detection of the detection species, for example, because it enables hybridization-based binding of the detection species at a specific location. In addition to facilitating high-speed specific detection, such physical attachment can enhance the use of the present method in the multiplex detection of several different target nucleic acids. In an embodiment, the second oligonucleotide probe is attached to a nucleic acid lateral flow strip or electrochemical probe, a 96-well plate, a bead, or an array surface. Thus, at least one species within the amplification product is localized to the physical location of the second oligonucleotide probe, which is easily detected after the formation of the detection species at that location. Alternatively, it may be advantageous to use a single-stranded oligonucleotide as the moiety attached to the second oligonucleotide probe to enable attachment to a solid material. In this way, the sequence of the solid-phase attached oligonucleotide can be independently defined as the target nucleic acid sequence to improve binding efficiency. Thus, in an embodiment, the moiety enabling attachment of the second oligonucleotide probe to the solid material is a single-stranded oligonucleotide. The single-stranded oligonucleotide may be designed to have improved affinity and hybridization efficiency to enhance the performance of the present method. For example, in a specific embodiment of the present method, a separate oligonucleotide having a sequence optimized for strip-based hybridization is used, which can efficiently hybridize to a single-stranded oligonucleotide moiety present in the second oligonucleotide probe rather than directly attaching the second oligonucleotide probe to the lateral flow strip.

[0122] In various studies, the inventors have significantly improved the performance of the present method by nucleic acid lateral flow using a single-stranded oligonucleotide as the attachment moiety of a second oligonucleotide probe, which provides an enhanced strip-phase hybridization sequence. For example, a GC-rich sequence may be used for strip-phase hybridization, or a longer sequence having a higher Tm may be used, which supplements the length of the second oligonucleotide probe. Alternatively, the single-stranded oligonucleotide moiety may include one or more modified base or nucleotide-nucleotide linkages, such as PNA, LNA, or G-clamp, to enhance its affinity. It has been observed that when a repeat sequence motif is used in the single-stranded oligonucleotide moiety, a surprising improvement in hybridization efficiency is observed that is not predicted by the predicted Tm. Accordingly, in the embodiments, the sequence of the single-stranded oligonucleotide moiety comprises three or more repeat copies of a 2 to 4 base DNA sequence motif. For example, in various studies using such sequence motifs, the inventors observed a significant improvement in detection responsiveness due to nucleic acid lateral flow, often with a signal enhancement of more than 100 times.

[0123] Accordingly, in an embodiment where the presence of a detection species is detected by nucleic acid lateral flow, the nucleic acid lateral flow uses one or more nucleic acids that enable sequence-specific hybridization of a moiety that enables attachment of a second oligonucleotide probe and a solid material.

[0124] Additional advantages are imparted by separating the target nucleic acid sequence from the solid material or the detection means for attachment, which may be made possible by the use of a single-stranded oligonucleotide as the detection moiety within the first oligonucleotide probe and / or as the attachment moiety with the second oligonucleotide probe. In this way, the device and / or detection means comprising the relevant solid material for attachment or the solid material, such as a nucleic acid lateral flow strip, can be optimized and defined regardless of the sequence of the target nucleic acid being detected. Such "universal" detection devices can be used according to the application and target without modification. For example, a nucleic acid lateral flow strip having lines printed on it corresponding to a set of commercially available oligonucleotide sequences that have efficient strip-phase hybridization capabilities and no unintended leakage signals can be defined, optimized, and efficiently manufactured independently of the development of the oligonucleotide primers and probes of the present method for the detection of multiple target nucleic acid sequences.

[0125] In many embodiments, detection may be performed in a quantitative manner. Thus, the level of single-stranded target nucleic acid in the sample may be quantified in step c). Quantification may be performed, for example, by colorimetrically, fluorescently, or electrically measuring the detection species over the time course of the reaction at multiple time points in addition to a single endpoint. Alternative strategies for quantification include sequential dilution of the sample similar to droplet digital PCR. In additional embodiments, the level of single-stranded target nucleic acid in the sample may be measured semi-quantitatively. For example, when the intensity of the colorimetric signal on a nucleic acid lateral flow strip corresponds to the approximate level of single-stranded target nucleic acid in the sample. Alternatively, an inhibitor may be used in which the number of copies of the single-stranded nucleic acid target must exceed a predetermined specific number to overcome the inhibitor and produce a detectable number of copies of the detection species.

[0126] In the method of the present invention, the second oligonucleotide probe is attached to a solid material or a moiety that enables attachment to the solid material. Optionally, in the embodiments, one or more other oligonucleotide primers and probes may also be attached to a solid material or a moiety that enables attachment to the solid material. It will be apparent to those skilled in the art that attaching oligonucleotides to solid materials can be achieved in various other ways. For example, a number of different solid materials are available that have a sufficient density of functional groups or are capable of attachment or functionalization to be useful for the purpose of attaching or reacting a suitably modified oligonucleotide probe. Additionally, these solid materials of various shapes, sizes, and forms are available, including beads, resins, surface-coated plates, slides, and capillaries. Examples of such solid materials used for the covalent attachment of oligonucleotides include, without limitation, glass slides, glass beads, ferrite core polymer-coated magnetic microbeads, silica microparticles or magnetic silica microparticles, silica-based capillary microtubes, 3D reactive polymer slides, microplate wells, polystyrene beads, poly(lactic) acid (PLA) particles, poly(methyl methacrylate) (PMMA) microparticles, controlled porous glass resins, graphene oxide (graphen) surfaces, and functionalized agarose or polyacrylamide surfaces. Polymers such as polyacrylamide have the additional advantage that functionalized oligonucleotides can be covalently bonded during the polymerization reaction between the monomers (e.g., acrylamide monomers) used to produce the polymer. Functionalized oligonucleotides are included in the polymerization reaction to produce a solid polymer containing covalently bonded oligonucleotides.This polymerization represents a highly efficient means of attaching oligonucleotides to solid materials by controlling the size, shape, and form of the resulting oligonucleotide-attached solid material.

[0127] Generally, to attach oligonucleotide probes to these solid materials, oligonucleotides having functional groups at the 3' or 5' ends are synthesized; however, functional groups can also be added during the oligonucleotide generation process at almost any base position. Subsequently, a specific reaction is performed between the functional group(s) within the oligonucleotide and the functional group on the associated solid material to form a stable covalent bond, thereby producing an oligonucleotide attached to the solid material. Typically, these oligonucleotides are attached to the solid material by the 5' or 3' ends. For example, two commonly used and reliable attachment chemicals utilize thiol (SH) or amine (NH3) groups and functional groups within the oligonucleotide. The thiol group can react with the maleimide moiety on the solid support to form a thioester bond, while the amine can react with the modified carboxylic acid succinimidyl ester (NHS ester) to form an amide bond. Several other chemicals may also be used. In addition to chemically conjugating the oligonucleotide probe to a solid material, it is possible and advantageous to directly synthesize the oligonucleotide probe on the solid material for use in carrying out the present method.

[0128] In another embodiment, the second oligonucleotide probe is attached to a moiety that enables attachment to a solid material. One strategy is to use an affinity binding method in which a moiety enabling specific binding is attached to the oligonucleotide probe to facilitate attachment to a related affinity ligand. This can be accomplished by using an affinity tag, such as an antibody-antigen binding or a poly-histidine tag, for example, or by using nucleic acid-based hybridization in which a complementary nucleic acid is attached to a solid material, for example, a nitrocellulose nucleic acid lateral flow strip. An exemplary such moiety is biotin, which itself can form a high affinity binding to streptavidin or avidin attached to a bead or other solid surface.

[0129] The presence of two or more different target nucleic acids of a given sequence can be detected in the same sample. In an embodiment of the method, a separate series of steps a), b), and c) are performed using different oligonucleotide primers and oligonucleotide probes for each of the two or more target nucleic acids, and the separate steps may be performed simultaneously. For example, in an embodiment, one set of oligonucleotide primers and oligonucleotide probes will be used for the detection of one target nucleic acid in the sample, and another set of oligonucleotide primers and oligonucleotide probes will be used for the detection of another target nucleic acid in the same sample. The detection of detection species generated from two or more different sets of primers / probes can be coupled to specific signals, such as different colorimetric or fluorescent dyes or enzymes, respectively, to enable multiplex detection. Alternatively, multiplex detection can be achieved by attaching a second oligonucleotide probe to a solid material directly or indirectly through a moiety that enables attachment to the solid material. This approach utilizes the physical separation of detection species generated by a different series of steps a), b), and c) rather than relying on other detection means. Thus, for example, a single dye can be used in a nucleic acid lateral flow to detect multiple different target nucleic acids, each generated different detection species is localized to a specific printing line of the lateral flow strip, and direct or indirect sequence-based hybridization to a second oligonucleotide probe forms the basis for differential detection. Alternatively, an electrodetection array may be used in which multiple detection species are localized to individual regions of the array through hybridization in a multiple reaction where multiple different second oligonucleotide probes are attached to specific regions of the array and thus multiple different detection species are generated simultaneously.

[0130] The aforementioned detection processes, such as nucleic acid lateral flow and electrical detection, and the ability to easily detect multiple different target nucleic acids within the same sample are made possible by the essential requirements of the present method for two oligonucleotide probes. Thus, this strongly demonstrates the advantages of the method of the present invention compared to known methods.

[0131] The present invention is widely useful in various fields and applications where the detection of a target nucleic acid of a specific sequence within a sample is required. This provides a simple, low-cost, and high-speed means of measuring the presence of a target nucleic acid sequence within a sample. By listing the fields of application without limitation, the inventors believe that the invention may be valuable in fields such as diagnostics, forensics, agriculture, animal health, environment, defense, human genetic testing, prenatal testing, blood contamination testing, pharmacogenomics or pharmacokinetics and microbiology, and clinical and biomedical research. Suitably, the sample is a biological sample, such as a human sample. The sample may be a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample.

[0132] The detection of target nucleic acids is for the diagnosis, prognosis, or monitoring of diseases or disease states such as infectious diseases 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, gastric cancer, or melanoma, or but not limited to HIV, influenza, RSV, rhinovirus, norovirus, tuberculosis, HPV, meningitis, hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, bubonic plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox, or cancer, or for human genetic testing, prenatal testing, blood contamination screening, pharmacogenetics, or It can be used in the field of pharmacokinetics.

[0133] The present invention may be used with an array of various types of samples, such as, for example, nasal swabs or adsorbates, nasopharyngeal swabs or adsorbates, throat swabs or adsorbates, cheek swabs or adsorbates, blood or samples derived from blood, urine or samples derived from urine, sputum or samples derived from sputum, feces or samples derived from feces, cerebrospinal fluid (CSF) or samples derived from CSF, and gastric fluid or samples derived from gastric fluid, and human or animal samples derived from any form of tissue biopsy or body fluid. In addition, the inventors performed the present method on a wide range of samples comprising at least 10 to 20% of clinical specimens: nasal swabs of VTM, nasopharyngeal swabs of VTM, low-viscosity prepared medium, throat swabs of liquid Amies, HSV wound swabs of M4 medium, synovial fluid, sputum treated with DNA capture beads after 2M NaOH / isopropanol, rectal swabs of TE, fecal samples treated with homogenized DNA capture beads, CSF, APTIMA swabs, amniotic fluid, oral swabs of liquid Amies, urine, VRE swabs of TE, pleural fluid, whole blood, K2EDTA plasma, L. heparin plasma, and serum. These experiments demonstrated the remarkable versatility of the present method for various clinical applications and the absence of inhibition observed in the relevant samples. This is completely different from other methods inhibited by inhibitors observed in biological specimens, such as heparin and phytic acid that inhibit PCR, and thus demonstrates the possibility of using this method on low-cost or single-use devices without any requirements for complex sample preparation procedures.

[0134] The target nucleic acid may be, in particular, (a) a virus or derived from viral nucleic acid material, (b) a bacterium or derived from bacterial nucleic acid material, (c) circulating cell-free DNA released from cancer cells, (d) circulating cell-free DNA released from fetal cells, or (e) microRNA or derived from microRNA.

[0135] The single-stranded target nucleic acid for the present method may be of natural or non-natural origin. The target nucleic acid may be generated in situ prior to the performance of the present method or generated from naturally occurring nucleic acids. The single-stranded target nucleic acid for the present method may be prepared by one or more additional steps performed prior to or concurrently with step a), and the additional steps may include one or more enzymes such as polymerases and restriction enzymes. The generation of the target nucleic acid for the present method in this manner has many possible advantages, such as enabling much higher-level multiplexing analysis and / or overcoming initial background. For example, highly specific transformation of nucleic acid material in biological samples may be performed without an amplification step prior to amplification in step a). Samples may be processed for the present method, for example, containing one or more modified dNTPs, purified, subjected to buffer exchange, subjected to exome capture, partially depleted of contaminants, and / or converted into single-stranded target nucleic acid. If the "actual" target nucleic acid of the detected sample is converted into a "surrogate" target nucleic acid for the performance of the present method by a reliable conversion (which may be less than 1:1, 1:1, or greater than 1:1, i.e., including some amplification element if possible), then the "actual" nucleic acid may be detected and / or quantified by the detection of the "surrogate" target nucleic acid. Furthermore, the generation of a surrogate target from a naturally occurring target in this manner may be used to specifically generate the target nucleic acid of the present method having any suitable sequence. In an embodiment where the single-stranded target nucleic acid is derived, for example, after the dissociation of two strands of double-stranded DNA by strand penetration, two complementary single-stranded nucleic acid targets may be present and may be amplified and detected in a mutual process by the same oligonucleotide primer and probe.If the target is the genome of a -ve strand single-strand RNA virus, a +ve strand transcript may also be present in the sample, and either one strand or both strands can be amplified and detected as a single-strand target nucleic acid in this method using the same oligonucleotide primer and probe.

[0136] Furthermore, the present invention is considered to have potential utility in screening samples for epigenetic modifications, such as CpG methylation of DNA sequences, and cell-free DNA. Such epigenetic modifications of specific cancer-related target genes can serve as useful biomarkers for numerous diseases and disease states. As awareness of the importance of epigenetic modifications in human diseases increases, the present invention can be used to specifically evaluate epigenetic modifications of specific target nucleic acid biomarkers based on the differential activity of strand-alternating DNA polymerases and / or restriction enzymes. Accordingly, in the embodiments, the target nucleic acid contains a site of epigenetic modification, such as methylation. Alternatively, as described above, the "actual" nucleic acid used to generate the "surrogate" target nucleic acid for performing the method contains a site of epigenetic modification.

[0137] A further aspect of the present invention relates to a kit for use in detecting nucleic acids of a predetermined sequence in a sample. Accordingly, the present invention also

[0138] a) a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence, a cleavage site, and a first hybridization sequence within a single-stranded target nucleic acid of a predetermined sequence in the 5' to 3' direction, and the second primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence, a cleavage site, and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence within the target nucleic acid in the 5' to 3' direction;

[0139] b) a first restriction enzyme that is not a nick-forming enzyme but can cleave a cleavage site by recognizing the recognition sequence of a first primer, and a second restriction enzyme that is not a nick-forming enzyme but can cleave a cleavage site by recognizing the recognition sequence of a second primer;

[0140] c) strand replacement DNA polymerase;

[0141] d) dNTP;

[0142] e) One or more modified dNTPs;

[0143] f) a first oligonucleotide probe attached to a moiety capable of hybridizing to a first single-stranded detection sequence of at least one species in an amplification product generated in the presence of the target nucleic acid and enabling the detection thereof; and

[0144] g) A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence of at least one species in the amplification product, and attached to a solid material or a moiety that enables attachment to a solid material.

[0145] Provides a kit including

[0146] In an embodiment, one of the first and second oligonucleotide probes of the kit is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme due to, for example, one or more sequence mismatches and / or one or more modifications, such as the presence of phosphorothioate linkages.

[0147] In an embodiment, one of the first and second oligonucleotide probes of the kit has five or more bases that are complementary to the hybridization region or the anti-complement of the first or second primer.

[0148] In another embodiment, the first oligonucleotide probe of the kit has some complementarity, for example, of five or more bases, with respect to the hybridization region of one of the first and second oligonucleotide primers, and / or the second oligonucleotide probe of the kit has some complementarity, for example, of five or more bases, with respect to the inverse complement of the hybridization region of the other of the first and second oligonucleotide primers.

[0149] In additional embodiments, the first and / or second oligonucleotide probes may have some complementarity or inverse complementarity with respect to the gap between the first and second hybridization sequences in the target nucleic acid as described above.

[0150] The kit may also contain reverse transcriptase.

[0151] The kit may further include means for detecting the presence of a detection species generated in the presence of a target nucleic acid. For example, the kit may further include a nucleic acid lateral flow strip, an electrochemical probe 96-well plate, a bead or array surface, and / or a colorimetric or fluorescent dye and / or a device for detecting a change in an electrical signal, and / or carbon or gold.

[0152] In various embodiments, the target nucleic acid and kit components included in the kit, e.g., 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 dNTP and / or one or more modified dNTPs and / or the first oligonucleotide probe and / or the second oligonucleotide probe and / or the first or second single-strand detection sequence of at least one species in the amplification product, are as defined herein for the method of the present invention.For example, the kit may include, without limitation, any combination of the features of the components described herein as follows: one of the first and second oligonucleotide probes is optionally blocked from elongation by DNA polymerase at the 3' end due to the presence of one or more sequence mismatches and / or one or more modifications, e.g., phosphorothioate linkages, and cannot be cleaved by the first or second restriction enzyme; the first restriction enzyme and the second restriction enzyme are the same restriction enzyme; one or more modified dNTPs are alpha-thiol modified dNTPs; the moiety enabling detection of the first oligonucleotide probe is a moiety capable of being attached to a colorimetric or fluorescent dye or a colorimetric or fluorescent dye such as biotin; and the moiety enabling attachment of the second oligonucleotide probe to a solid material is optionally a single-stranded oligonucleotide comprising three or more repeat copies of a DNA sequence motif of 2 to 4 bases; The first and second oligonucleotide primers include, for example, a stabilization sequence of 5 or 6 base lengths upstream of the restriction enzyme recognition sequence and the cleavage site at the 5' end; the hybridization regions of the first and / or second oligonucleotide primers are 6 to 30, for example, 9 to 16 base lengths; the first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 or 0 to 6 bases, and in certain embodiments, are separated by 3 to 15 or 3 to 6 bases, for example, 5, 7, or 11 bases, or are overlapped by, for example, 1 to 2 bases.

[0153] The kit may include means for detecting the presence of a detection species generated in the presence of a target nucleic acid, such as a nucleic acid lateral flow strip. In further embodiments, the kit further includes a third and / or fourth oligonucleotide primer as described herein.

[0154] The kit may also include reagents such as reaction buffers, salts, for example, divalent metal ions, additives, and excipients.

[0155] The kit according to the present invention may be provided with instructions for performing the method according to the present invention.

[0156] The present invention also provides a use of the kit of the present invention for detecting a single-stranded target nucleic acid of a predetermined sequence in a sample.

[0157] All optional and / or preferred embodiments of the present invention described herein in relation to the method of the present invention should also be understood to apply in relation to the kit of the present invention and its use, and vice versa.

[0158] As described above, the method and kit of the present invention are ideally suited for use in devices such as single-use diagnostic devices. Accordingly, the present invention also provides a device comprising a kit such as described above, particularly a kit comprising means for detecting the presence of a detection species generated in the presence of a target nucleic acid, such as a nucleic acid lateral flow strip. The device may be a powered device, e.g., an electric power supply, and may also include heating means, and may be a self-contained device, i.e., a device that does not require an auxiliary test device.

[0159] The method of the present invention may also be used independently of detection step c) to amplify a nucleic acid signal from a target nucleic acid of a predetermined sequence, and such method may be used, for example, when the amplified signal is stored and / or transported for detection of the target nucleic acid at a future date and / or, if necessary, at an alternative location. The amplified signal includes a detection species or a prior detection species generated through the performance of the method. Accordingly, in a further embodiment, the present invention provides a method for amplifying a nucleic acid signal from a target nucleic acid of a predetermined sequence in a sample comprising all or part of steps a) and b) of the method of the present invention, for example, part i or ii.

[0160] The present invention also provides a use of the kit of the present invention for amplifying a nucleic acid signal from a target nucleic acid of a predetermined sequence as defined above.

[0161] All optional and / or preferred embodiments of the present invention described herein in relation to the method of the present invention for detecting the presence of a target nucleic acid of a predetermined sequence in a sample should also be understood to apply in relation to a method for amplifying a nucleic acid signal from a target nucleic acid of a predetermined sequence.

[0162] The following examples serve to further illustrate various aspects and embodiments of the method described herein. These examples should not be construed as limiting in any way.

[0163] Examples

[0164] Materials and Methods

[0165] Unless otherwise indicated, the following materials and methods are used in the following examples.

[0166] Oligonucleotides: Unless otherwise indicated, custom oligonucleotides were prepared using the phosphoramidite method of the incorporated DNA technology.

[0167] Nucleic acid lateral flow: Carbon nanoparticles were conjugated to various biotin-binding proteins, e.g., streptavidin, via non-covalent adsorption. Typically, a colloidal carbon suspension was prepared in borate buffer and then sonicated using a probe sonicator. The carbon was then incubated at room temperature to adsorb onto the biotin-binding proteins. The carbon was used directly in the reaction mixture or applied to a glass fiber conjugate pad. Lateral flow strips were prepared by combining a sample pad, a nitrocellulose membrane, and an adsorbent pad (Merck Millipore) with a conjugate pad containing freeze-dried sugars and additives used to improve visual appearance, in accordance with the manufacturer's instructions. Before use in the lateral flow strips, relevant oligonucleotide(s) containing the reverse complement of the sequence to be detected in this method were printed onto the nitrocellulose membrane at specific locations and attached to the membrane via UV crosslinking.

[0168] Example 1

[0169] Performing a method of attaching a second oligonucleotide probe to a solid material nitrocellulose lateral flow strip

[0170] This embodiment describes a method of attaching a second oligonucleotide probe to a solid material, a nitrocellulose lateral flow strip, and not bringing the first oligonucleotide probe into contact with a sample simultaneously with the performance of amplification step a).

[0171] A first oligonucleotide primer was designed with a total length of 24 bases, comprising a stabilization region of 7 bases in the 5' to 3' direction; 5 bases of a recognition sequence for a restriction enzyme that is not a nick-forming enzyme; and a 12-base hybridization region containing the reverse complement sequence of the first hybridization sequence in the target nucleic acid. A second oligonucleotide primer was designed, comprising the same stabilization region and restriction enzyme recognition sequence, but including a 12-base hybridization region capable of hybridizing to the reverse complement of the second hybridization sequence in the target nucleic acid. In this embodiment, the first restriction enzyme and the second restriction enzyme are the same restriction enzyme. The restriction enzyme is an asymmetric double-strand cleavage restriction enzyme having an upstrand cleavage site downstream of the 5-base recognition sequence. The first and second hybridization sequences of the target nucleic acid are separated by 1 base.

[0172] An oligonucleotide primer was designed using a target nucleic acid such that the nucleotide base downstream of the cleavage site in the primer's anti-complement is adenosine, and alpha-thiol dATP is used as the modified dNTP in this method. The phosphothioate modification is inserted by a strand replacement polymerase to block the cleavage of the anti-complement strand.

[0173] A first oligonucleotide probe of a total length of 20 bases was designed, comprising a 12-base region complementary to at least one species in the amplification product in the 5' to 3' direction; a neutral spacer region of 6 bases; and a 3' biotin modification added during synthesis, said biotin modification enabling attachment of the first oligonucleotide probe and a colorimetric dye carbon nanoparticle. Carbon adsorbed to a biotin-binding protein was prepared and saturated with the first oligonucleotide probe. A second oligonucleotide probe of a total length of 49 bases was designed, comprising a neutral spacer containing 10 X thymidine bases in the 5' to 3' direction; and a 3X repeat of a 13-base region capable of hybridizing to a second single-strand detection sequence downstream of the first single-strand detection sequence in the at least one species in the amplification product. Approximately 30 pmol of said second oligonucleotide probe was printed on a nucleic acid flow strip.

[0174] A reaction was prepared comprising 1.6 pmol of first primer; 0.1 pmol of second primer; 250 μM 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) Sp-isomer (Sp-dATP-α-S) from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand replacement DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at various levels (++ = 1 amol, + = 10 zmol, NTC = no target control) to a total reaction volume of 10 μl in appropriate reaction buffer. The reaction was incubated at 45°C for 7 or 10 minutes. 6.5 μl of the termination reaction mixture was then added at 0.056 mg / ml -1 After adding 60 μl of lateral flow driving buffer containing the conjugated carbon, the second oligonucleotide probe was loaded onto a nucleic acid lateral flow strip attached to a printed line.

[0175] Figure 5 shows a photograph of a lateral flow strip obtained during the execution of the example. The arrow indicates the location where a positive signal appears after the second oligonucleotide probe is printed on the nitrocellulose strip. A clear black line corresponding to the presence of a carbon signal was observed only in the presence of the target nucleic acid at both target levels and at both time points, indicating rapid and responsive detection of the target nucleic acid sequence by the method of the present invention.

[0176] Example 2

[0177] The first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme, and the method of contacting the sample in step a) is performed.

[0178] This embodiment describes the execution of an embodiment of a method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and contacts the sample simultaneously with the execution of step a). In this embodiment, the inventors did not observe any significant inhibition of the amplification rate, which indicates that the pre-detection species accumulate in real time without interfering with the optimal cyclic amplification process. Not only was no inhibitory effect on the amplification process observed in the above embodiment, but the inventors also observed a remarkable improvement in the generated signal corresponding to at least a 100-fold increase in the amount of the detection species.

[0179] Example 2.1: A variation of the assay used in Example 1 was designed using an embodiment of a method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a). The same oligonucleotide primer, restriction enzyme, dNTP, modified dNTP, and polymerase used in Example 1 were used, but an alternative first oligonucleotide probe of a total length of 21 bases was designed, comprising a 5' biotin modification in the 5' to 3' direction; a neutral region of 8 bases; a 13 base region capable of hybridizing to at least one species in the amplification product; and a 3' phosphate modification, wherein the biotin modification enables the attachment of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the phosphate modification blocks its elongation by strand-replacement DNA polymerase. Carbon adsorbed to biotin-binding protein was prepared and saturated with the first oligonucleotide probe.

[0180] An alternative second oligonucleotide probe was designed comprising: a 14-base region capable of hybridizing to a second single-strand detection sequence upstream of a first single-strand detection sequence of at least one species in the amplification product in the 5' to 3' direction; a 6-base neutral spacer sequence; a repeat of the 14-base hybridization region; a second 6-base neutral spacer sequence; and a 10 X thymidine base spacer. Approximately 30 pmol of the second oligonucleotide probe was printed on a nucleic acid flow strip.

[0181] A reaction was prepared comprising 0.8 pmol of the first primer; 0.8 pmol of the second primer; 0.6 pmol of the first oligonucleotide probe; 300 μM Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand replacement DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at various levels (++ = 1 amol, + = 10 zmol, NTC = no target control) to a total reaction volume of 10 μl in appropriate reaction buffer. The reaction was incubated at 45°C for 6 minutes. 5 μl of the termination reaction mixture was then added to 0.03 mg / ml -1 After adding 60 μl of lateral flow drive buffer containing the conjugated carbon, it was loaded onto a nucleic acid lateral flow strip. A control reaction in which the first oligonucleotide probe was absent during the reaction was performed to demonstrate that no detection species were produced. To control for any unintended effects of the probe's presence during lateral flow strip detection, an equal level (0.6 pmol) of the probe was added to the control after step a).

[0182] Figure 6a provides a photograph of the nucleic acid lateral flow strip after development. A clear signal corresponding to the deposition of carbon nanoparticles was observed at both target levels when the first oligonucleotide probe was provided during the reaction. As expected, no signal was detected at the target levels when the first oligonucleotide was not provided during the reaction. This experiment clearly demonstrates the potential to substantially improve the generation of detected species in an embodiment of the method in which the first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by the first or second restriction enzyme, and comes into contact with the sample simultaneously with the performance of step a). It should be noted that, unlike Example 1, the first and second oligonucleotide primers at the same concentration are provided, allowing for more rapid amplification.

[0183] Example 2.2: To demonstrate the diversity of the above embodiments of the method using completely different target nucleic acids, a separate analysis was designed below. Oligonucleotide primers and oligonucleotide probes were designed for the relevant target nucleic acid, single-stranded DNA, in a manner similar to that described in Examples 1 and 2.1.

[0184] A reaction was prepared comprising 0.8 pmol of the first primer; 0.4 pmol of the second primer; 0.6 pmol of the first oligonucleotide probe; 300 μM Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand replacement DNA polymerase. Nucleic acid targets (single-stranded DNA targets) were added at varying levels (+ = 1 amol, NTC = no-target control) to a total reaction volume of 10 μl in appropriate reaction buffer. The reaction was incubated at 45°C for 6 minutes. 5 μl of the termination reaction mixture was then added to 0.08 mg / ml -1After adding 60 μl of lateral flow driving buffer containing the conjugated carbon, it was loaded onto a nucleic acid lateral flow strip. A control reaction was performed simultaneously with the execution of step a) involving a cleaved variant of the first oligonucleotide probe in contact with the sample.

[0185] FIG. 6b provides a photograph of the nucleic acid lateral flow strip after expansion. A clear positive signal is observable in the presence of the target nucleic acid and is not observed in the control group where the target is absent, which indicates the strong potential of the embodiment of the method in which the first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by the first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a), as well as the correct design and function of the analysis. As expected, only a very minimal signal was observed in the control group analysis using the cleaved form of the first oligonucleotide probe, which demonstrates the requirement for accurate hybridization of the first oligonucleotide probe simultaneously with the amplification in step a) for the generation of an efficient detection species.

[0186] Example 3

[0187] Performance of a method in which the presence of two or more different target nucleic acids of a specified sequence is detected in the same sample

[0188] This example demonstrates the possibility of a method for detecting two or more different target nucleic acids of a given sequence in a sample. Using two oligonucleotide probes in addition to the primers of the method provides an incorporation approach for detecting amplification products in a method ideally suited for detecting two or more different target nucleic acids in the same sample. In this example, the ability to differentially detect alternative detection species based on the sequence-specific hybridization of the second oligonucleotide probe was demonstrated.

[0189] First, to demonstrate the capability of a method used for the detection of two or more different target nucleic acids, the inventors developed a set of oligonucleotide primers and probes suitable for the detection of two distinct targets (A and B). In each case, a first oligonucleotide probe was designed comprising a 5' biotin modification in the 5'-to-3' direction, a 7-base stabilization region, a 5-base restriction endonuclease recognition site, a region complementary to the 3' end of target A or B including a phosphorothioate binding to a restriction enzyme cleavage site of 11 to 13 bases, and a 3' phosphate modification. A second oligonucleotide probe was designed comprising a 12 to 14 base region complementary to the 5' end of target A or B in the 5' to 3' direction, a neutral spacer of 5 X thymidine bases, and a 12-base single-stranded oligonucleotide moiety as a moiety enabling attachment of the second oligonucleotide probe to a solid material. Sequences of single-stranded oligonucleotide attachment moietys for each target were designed using different sequences to enable attachment of each detection species to different locations on a lateral flow strip. A nucleic acid lateral flow strip was prepared containing individual spots of 30 pmol of oligonucleotides, each containing an inverse complement sequence for the respective single-stranded oligonucleotide detection moiety at separated locations.

[0190] 0.032 mg / ml adsorbed to biotin-binding protein -1 A reaction was prepared containing 0.5 pmol of the first oligonucleotide probe for Target A and Target B; and 0.5 pmol of the second oligonucleotide primer for Target A and B in 65 μl of an appropriate carbon-containing buffer. Different levels of each target (+ = 0.1 pmol; ++ = 1 pmol) were added individually to the separate reactions, and the two targets were added together. A targetless control (NTC) was also performed.

[0191] Figure 7a shows a photograph of the lateral flow strip obtained in the experiment. A clear black spot corresponding to the deposition of carbon containing the detected species was observed at both target levels and in both analyses. Additionally, when the two reactions were performed simultaneously, signals corresponding to targets A and B were observed. No background signals or leakage signals were observed between the other analyses.

[0192] To demonstrate the robustness of the present method, additional experiments were conducted to develop three separate analyses to prove the feasibility of the method for detecting three different target nucleic acids of a given sequence in a sample. A methodology similar to that described above was used. Figure 7b shows a photograph of the acquired lateral flow strip. Targets P1, P2, and P3 were added individually and in various combinations as indicated. The reverse complement for the single-stranded oligonucleotide detection moiety of the second oligonucleotide probe was printed as a separate line on the nucleic acid lateral flow strip. The black signal indicates the deposition of carbon-attached detection species localized to the expected location in all cases for rapid and responsive detection without unintended leakage signals between the analysis and the background signal. An equivalent experiment involving four separate analyses demonstrated the feasibility of the method for detecting four different target nucleic acids (P1, P2, P3, and P4) of a given sequence in a sample, and the results are shown in Figure 7c. In these four-target experiments, P4 was present as a positive control for all reactions, and the other targets were added individually to each reaction. The photograph of the indicated lateral flow strip shows a clear black band at the expected location, which corresponds to the presence of the relevant detection species bound to the carbon. This multiple analysis demonstrates the potential of the method for use in diagnostic tests for diseases caused by various pathogens; the detection of the presence of the detection species in the control analysis indicates that the method has been successfully performed, and the visualization of one or more of the other detection species on the lateral flow strip indicates the presence of the relevant causative pathogen(s) in the appropriate clinical specimen. Although it is rare in diagnostic applications such as the field of infectious diseases to observe co-infections where more than one pathogen is present in the same specimen, the method of the present invention is highly versatile for any combination of targets in the multiple complex reactions detected. Fig. 7d shows the results of experiments with different combinations of the four targets (P1, P2, P3, and P4) added.The ability to detect each target individually and to detect three other targets when each target is missed without a non-specific background demonstrates the remarkable detection specificity of the method of the present invention.

[0193] In various other experiments described above, the inventors also performed multiplex analysis for the detection of 3 to 5 targets at very low target concentrations, e.g., 1 zmol (600 copies) or 17 ymol (10 copies). In this example, the inventors clearly demonstrated the feasibility of a method for detecting the presence of two or more different target nucleic acids of a given sequence in a sample, and the possibility of rapid and low-cost signal detection, for example, by nucleic acid lateral flow. The fact that two or more different target nucleic acids of a given sequence can be easily detected in the same sample is a unique and advantageous characteristic of the method of the present invention. For each additional target detected, an additional set of oligonucleotide primers is required, which presents a significant problem in detecting the presence of two or more different target nucleic acids in conventional methods without temperature cycling, because the additional primers lead to an increased tendency to form non-specific amplification products. In the method of the present invention, this problem is overcome by an improvement in specificity, such as that resulting from the use of modified bases, improved enzyme selection, and the formation of detection species using oligonucleotide probes that utilize additional sequence-specific hybridization events.

[0194] Example 4

[0195] Performing a method in which the first and second hybridization sequences of a target nucleic acid are separated into five bases

[0196] This embodiment describes the execution of a method in which first and second hybridization sequences in a target nucleic acid are separated by five bases. The ability to use a target-derived sequence that is not present in the oligonucleotide primer and is generated in a target-dependent manner only in the amplification product when two oligonucleotide primers are designed such that a gap exists between the first and second hybridization sequences provides the possibility for enhanced specificity in an embodiment of the method that can overcome any background signal arising from initial synthesis or primer-primer binding. In the above embodiment, sequence-specific hybridization of the first or second oligonucleotide probe is designed using the gap between the two hybridization regions so that only the detection species is generated when the amplification product contains the correct target-derived sequence.

[0197] In this embodiment, the inventors designed various analyses to demonstrate the hybridization of the second oligonucleotide probe into various different amplification products in which only the sequence of the gap between the first and second hybridization sequences within the target nucleic acid differs. The second oligonucleotide probe was designed to include an 11-base hybridization region for at least one species at the 5' end of the amplification product. The region consisted of a 5-base sequence of the inverse complement sequence for an additional target-derived sequence within the amplification product derived from the gap between the two primers and a 7-base sequence of the inverse complement sequence of the first oligonucleotide primer. The second oligonucleotide probe also includes a neutral spacer of 5X thymidine bases in the 5' to 3' direction and a 12-base single-stranded oligonucleotide moiety for attachment to a solid material. A nitrocellulose nucleic acid flow strip was prepared by printing 30 pmol of oligonucleotide of the inverse complement sequence of the moiety. The first oligonucleotide probe was designed to have the same sequence as the second oligonucleotide primer but to include a 5' biotin modification, a 3' phosphate modification, and a phosphorothioate nucleotide linkage at the location of the restriction enzyme cleavage site.

[0198] Four different artificial target nucleic acid sequences (T1, T2, T3, and T4) were designed, each having the exact sequence corresponding to the first and second hybridization sequences, but with a 5-base difference between the first and second hybridization sequences: T1 contains the exact base for detection that is fully complementary to the 11-base hybridization region of the second oligonucleotide probe; T2 contains a 4-base mismatch of the 5 bases in the gap; T3 was designed so that 4 bases of the 5 bases in the gap are removed, making the species of the amplification product 4 bases shorter; and T4 contains a 2-base mismatch of the 5 bases in the gap.

[0199] A reaction was prepared containing 3.6 pmol of first oligonucleotide primer; 1.8 pmol of second oligonucleotide primer; 2.4 pmol of first oligonucleotide probe; 300 μM Sp-dATP-α-S, 60 μM dTTP, dCTP, dGTP; 12 U restriction enzyme; and 12 U Bacillus strand replacement DNA polymerase, with a total reaction volume of 60 μl in appropriate reaction buffer. After adding 1 amol target (T1, T2, T3, or T4) to each reaction, the mixture was incubated at 45°C for 6.5 minutes, and then 53.5 μl of the 60 μl reaction was driven on a lateral flow strip. Before applying the reaction to the lateral flow strip, 1.5 pmol of second oligonucleotide probe and 2 μg of carbon adsorbed to biotin-binding protein were deposited on the conjugate pad and allowed to dry for 5 minutes.

[0200] Figure 8 shows photographs of nucleic acid lateral flow strips obtained in the experiment. The strip obtained with target T1 shows a clear black line corresponding to a carbon-attached detection species attached to the solid material of nitrocellulose, indicating that the assay developed in this example, including the oligonucleotide primer and probe, functions correctly and has the potential for rapid and responsive detection. In the reactions performed with targets T2 and T3, no carbon corresponding to a positive signal appeared, indicating that both the removal of four mismatches and four bases eliminate the ability of the second oligonucleotide to effectively hybridize to the pre-detection species generated in the reaction. A very faint signal was observed in the strip generated using T4, indicating that the presence of only two mismatched bases results in a significant loss of the ability of the second oligonucleotide probe to successfully hybridize to the pre-detection species and generate a detection species capable of binding to a line on the strip. Polyacrylamide gel electrophoresis was performed using repeated reactions to confirm that all reactions with all targets functioned correctly and produced a significant amount of amplification product. The expected size change was observed in the reaction performed with target T3 with 4 bases cleaved.

[0201] This example demonstrates a method in which the first and second oligonucleotide probes, which are essential features of the present invention, can be used not only to provide rapid and responsive detection of amplification products but also to provide additional target sequence-based specificity testing of amplification products beyond that resulting from primer hybridization alone. This powerful technique overcomes the known problems of prior art methods caused by non-target-specific background amplification in specific analyses resulting from initial synthesis or primer-primer binding. This indicates that the method of the present invention exhibits enhanced specificity compared to prior art methods while maintaining responsive detection and rapid, low-cost visualization of results.

[0202] Example 5

[0203] A method in which a moiety enabling the attachment of a second oligonucleotide probe to a solid material is an antigen, and a corresponding antibody is attached to a solid surface, a nitrocellulose lateral flow strip.

[0204] In the method of the present invention, a plurality of different moietyes may be used as moietyes for the attachment of the second oligonucleotide probe and the solid material. This embodiment indicates that a method may be performed in which a moiety enabling the attachment of the second oligonucleotide probe and the solid material is an antigen and a corresponding antibody is attached to a solid surface nitrocellulose lateral flow strip.

[0205] The second oligonucleotide probe was designed to contain a 32-base sequence including a homology region for at least one of the 3' digoxigenin NHS ester modifications and amplification products added during synthesis. A Fab fragment anti-digoxigenin antibody (Sigma-Aldrich) purified from sheep was immobilized on a nucleic acid lateral flow strip by spotting and air drying.

[0206] 0.016 mg / ml of various levels of target (+++ = 1 pmol; ++ = 0.1 pmol; + = 10 fmol; NTC = no target control) adsorbed to biotin-binding proteins -1The performance of the second oligonucleotide probe was demonstrated in an experiment in which it was added to 60 μl of a designed reaction buffer containing the reagents required for detection using a carbon nucleic acid lateral flow reaction containing carbon. A strip was prepared with 0.5 μg of anti-digoxigenin Fab fragments spotted on a strip in 0.2 μl of buffer containing 2.5 mM borate and 0.5% Tween 20. The solution was allowed to dry on the nitrocellulose membrane of the lateral flow strip for 2 hours. After incubating the reaction at 45°C for 2 minutes to form the designed detection species, the entire reaction mixture of each reaction was applied to the lateral flow strip.

[0207] Figure 9 shows a photograph of the lateral flow strip generated in the experiment. Black spots corresponding to carbon deposition in the lateral flow strip are observable at each target level but not at the NTC, indicating specific detection of the detection species. A combination of biotin-based affinity interactions for the attachment of the detection moiety (carbon) and antibody-based affinity interactions for the attachment moiety of the solid material was demonstrated. This example demonstrates the versatility of the method in terms of various approaches that can be used for the attachment of the second oligonucleotide probe to the solid material.

[0208] Example 6

[0209] A method for attaching a second oligonucleotide probe to a solid material, wherein the moiety enabling attachment to the solid material is a single-stranded oligonucleotide comprising four repeat copies of a three-base DNA sequence motif, and the inverse complement of the single-stranded oligonucleotide sequence is attached to the solid material.

[0210] This embodiment describes the execution of a method in which a single-stranded oligonucleotide comprising four repeat copies of a three-base DNA sequence motif is a moiety that enables attachment of a second oligonucleotide probe to a solid material. As described above, an embodiment of the method using a single-stranded oligonucleotide as the detection moiety of the second oligonucleotide probe presents a simple and versatile aspect of the present method, which facilitates detection by nucleic acid lateral flow and enables the detection of multiple different target nucleic acids in the same sample. Furthermore, by pre-defining the single-stranded oligonucleotide detection moiety and optimizing it for efficient strip-phase hybridization, detection sensitivity can be improved and efficient scale-up manufacturing of nucleic acid lateral flow strips can be provided.

[0211] In one aspect of the present invention, the inventors observed a remarkable improvement in strip-phase hybridization by using a single-stranded oligonucleotide detection moiety composed of multiple repeat copies of a DNA sequence motif. This example presents the results of multiple parallel experiments in which the performance of an analysis using a second oligonucleotide directly attached to a lateral flow strip is substantially improved by using a single-stranded detection moiety comprising four repeat copies of a three-base DNA sequence motif, and the inverse complement of said single-stranded oligonucleotide sequence is attached to the lateral flow strip.

[0212] Example 6.1: An analysis was designed using an embodiment of a method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a). A first oligonucleotide probe was designed with a total length of 25 bases, comprising: a 5' biotin modification in the 5'-to-3' direction; a neutral region of 7 bases; 5 bases of a restriction enzyme recognition site that is not a nick-forming enzyme; a 13-base region capable of hybridizing to a first hybridization region of the target containing a phosphothioate binding to a cleavage site for the restriction enzyme; and a 3' phosphate modification, wherein the biotin modification enables the attachment of the first oligonucleotide probe to a colorimetric dye and carbon nanoparticles, and the phosphate modification blocks its elongation by strand-replacement DNA polymerase.

[0213] Two alternative second oligonucleotide probes were designed to detect the same target species (I and II). A second oligonucleotide probe 'I' was designed containing 3X repeats of a 14-base region capable of hybridizing to the inverse complement of the second hybridization sequence in the target in the 5' to 3' direction; and 9X thymidine base spacers. A nucleic acid lateral flow strip was prepared with a spot containing 30 pmol of the probe.

[0214] An alternative second oligonucleotide probe 'II' was designed comprising: a 14-base region capable of hybridizing to the inverse complement of the second hybridization region in the 5' to 3' direction from the target; a neutral spacer of 5 X thymidine bases; and a single-stranded oligonucleotide moiety of 12 bases comprising a 4X repeat of a 3-base sequence motif acting as a moiety to enable attachment of the second oligonucleotide probe to a solid material.

[0215] An additional single-stranded oligonucleotide was designed comprising an 11 X thymidine base spacer in the 5' to 3' direction; and a 36-base region comprising a 12 X repeat of an inverse complement to a 3-base sequence motif that forms a moiety enabling attachment of the solid material and the second oligonucleotide II. For the second oligonucleotide probe II, a nucleic acid lateral flow strip was prepared by spotting 30 pmol of the additional single-stranded oligonucleotide.

[0216] 0.016 mg / ml adsorbed to biotin-binding protein -1 Reactions were performed to test the performance of Oligonucleotide Probes I and II, containing 0.5 pmol of the first oligonucleotide probe in 60 μl of an appropriate carbon-containing buffer. A reaction for II was prepared in the same manner, but with the addition of 0.5 pmol of the second oligonucleotide probe II. Nucleic acid targets (single-stranded DNA targets representing at least one species in the amplification product generated from the designed assay reagents) were added at varying levels (+++ = 1 pmol, ++ = 0.1 pmol, NTC = no target control). After incubating the prepared reactions at 45°C for 2 minutes, the entire reaction mixture was loaded onto an appropriate nucleic acid lateral flow strip.

[0217] Figure 10a shows a photograph of the lateral flow strip obtained in the experiment, with the left panel displaying the results for the second oligonucleotide probe I and the right panel displaying the results for the second oligonucleotide probe II. Black spots corresponding to the deposition of carbon-attached detection species were visualized in the presence of the target. For the second oligonucleotide probe II containing a repeat sequence motif, a stronger signal was observed at all target levels.

[0218] Example 6.2: To demonstrate the versatility of the above embodiments of the method and its broad applicability, separate analyses were subsequently designed for completely different target nucleic acids. Oligonucleotide probes were designed for the relevant target nucleic acid, single-stranded DNA, in a manner similar to that described in Example 6.1, using two versions of the second oligonucleotide probe, again designated as 'I' and 'II', and various target levels (+++ = 1 pmol, ++ = 0.1 pmol, + = 0.001 pmol). A much more surprising effect was observed, as indicated in the photograph of the generated lateral flow strip shown in Fig. 10b. The second oligonucleotide probe I, tested at the two lower target levels, produced no signal, while the corresponding repeat sequence oligonucleotide probe II produced a clear positive signal, indicated by a black spot of deposited carbon.

[0219] This embodiment demonstrates a significant improvement to lateral flow hybridization-based detection using a second oligonucleotide detection moiety comprising a repeat copy of a DNA sequence motif. This demonstrates that a 100-fold improvement in the responsiveness of nucleic acid lateral flow-based detection of detection species can be achieved. Signal intensity is enhanced and signals occur more rapidly, indicating the potential for the above embodiment of the invention to be easily applied to applications involving rapid detection, such as nucleic acid lateral flow. Additionally, the possibility of using a single-stranded oligonucleotide as a detection moiety attached to the second oligonucleotide probe is exemplified.

[0220] Example 7

[0221] Use of RNA virus detection methods in clinical specimens

[0222] This embodiment demonstrates the performance of a method for detecting RNA viruses in clinical specimens using an embodiment in which a first oligonucleotide probe is brought into contact with a sample simultaneously with the performance of amplification step a), a second oligonucleotide probe and a moiety enabling attachment to a solid material is a single-stranded oligonucleotide comprising four repeat copies of a three-base DNA sequence motif, and an inverse complement of said single-stranded oligonucleotide sequence is attached to a solid material. In various investigations, the inventors have typically detected very low copies of RNA targets, such as viral genome extracts. For example, using quantified viral genome extracts, the inventors used the method of the present invention to detect fewer than 100 genomic equivalent copies of the virus within a total of 10 minutes by amplification step a) of less than 5 minutes. This remarkable speed and responsiveness demonstrate the potential for applying the present method to the field of diagnostics. Accordingly, in this embodiment, the inventors developed an assay for detecting pathogenic single-stranded RNA viruses and demonstrated the performance of the assay using clinical specimens infected with the virus.

[0223] A first oligonucleotide primer of a total length of 25 nucleotides was designed, comprising: an 8-base stabilization region synthesized to include phosphorothioate bonds between each base in the 5' to 3' direction; 5 bases of a recognition site for a restriction enzyme that is not a nick-forming enzyme; and a 12-base hybridization region containing the reverse complement sequence of the first hybridization sequence in the target nucleic acid designed to target a region within the single-stranded RNA virus genome. A second oligonucleotide primer was designed, comprising a restriction enzyme recognition sequence that includes the same but phosphorothioate-free stabilization region and the same but 12-base hybridization region capable of hybridizing to the reverse complement of the second hybridization sequence. In this embodiment, the first restriction enzyme and the second restriction enzyme are the same restriction enzyme. The first and second hybridization sequences of the target nucleic acid are separated by 0 bases.

[0224] Oligonucleotide primers were designed using target nucleic acids such that the nucleotide base downstream of the cleavage site in the primer's anti-complement is adenosine, and alpha-thiol dATP is used as a modified dNTP for use in this method. The phosphothioate modification is inserted by strand replacement DNA polymerase or reverse transcriptase to block the cleavage of the anti-complement strand.

[0225] A first oligonucleotide probe of a total length of 24 bases was designed, comprising: a 5' biotin modification added during synthesis in the 5' to 3' direction, wherein the biotin modification enables the attachment of the first oligonucleotide probe and the colorimetric dye carbon nanoparticles, a stabilization region of 8 bases; 5 bases of a recognition sequence for a non-nick-forming restriction enzyme, wherein the cleavage site for the restriction enzyme in the first oligonucleotide probe is protected by a phosphothioate nucleotide linkage 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 elongation by strand replacement DNA polymerase.

[0226] A second oligonucleotide probe of a total length of 31 bases was designed, comprising: a 14-base region capable of hybridizing to a second single-strand detection sequence downstream of a first single-strand detection sequence of at least one species in the amplification product in the 5' to 3' direction; a spacer containing 5 X thymidine bases; 4 X repeats of a 3-base DNA sequence motif; and an anti-complement immobilized on a lateral flow strip. A neutral spacer containing 11 X thymidine bases was designed; and a fixed lateral flow printed oligonucleotide of a total length of 47 bases was designed, comprising 12 X repeats of a 3-base sequence motif complementary to the 3-base sequence motif of the second oligonucleotide probe. A 5 X triplicate repeat different from the second oligonucleotide probe in the 5' to 3' direction; A 20-base-long lateral flow control oligonucleotide was designed containing a neutral spacer with 5 X thymidine bases and a 3' biotin molecule added during synthesis. The control oligonucleotide was bound to the anti-complement of a lateral flow strip to verify a successful carbon lateral flow procedure.

[0227] A reaction was prepared comprising 1.8 pmol of first primer; 9.6 pmol of second primer; 3.6 pmol of first probe; 1 pmol of second probe; 300 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 28 U of restriction enzyme; 14 U of Bacillus strand replacement DNA polymerase; 35 U of viral reverse transcriptase; 3.5 U of RNaseH; and 3 μg of carbon adsorbed to biotin-binding protein. 5 μl of nasopharyngeal swab samples (supplied by Discovery Life Sciences) collected from patients in a clinical setting, including 7 virus-positive samples and 6 virus-negative clinical samples (verified by PCR analysis). The reaction was performed in a volume of 70 μl in appropriate reaction buffer. After incubating the reaction at 45°C for 4 minutes and 30 seconds, the entire reaction was loaded onto a nucleic acid lateral flow strip printed with an anti-complement for the control oligonucleotide (top line) and an anti-complement for the 3-base triplet repeat moiety of the second oligonucleotide probe at approximately 50 pmol (bottom).

[0228] Figure 11 shows a photograph of the lateral flow strip obtained during the execution of this example. Arrows confirm successful lateral flow driving and thus indicate the location of the counter-complement (CTL) for the control oligonucleotide appearing in both positive and negative analyses, and the location where the counter-complement for the triplicate repeat moiety of the second oligonucleotide probe is printed (+), thereby indicating the location where a positive signal appears. The upper panel (+ve) shows results obtained from virus-positive clinical samples, and the lower panel (-ve) shows results obtained from virus-negative samples. A clear black line indicating the presence of the target nucleic acid is present in each positive sample, demonstrating the rapid detection of clinical samples by the method of the present invention. No false positives were observed, indicating the complete absence of non-specific generation of the detected species, as is the case with initial synthesis or primer-primer binding. No false negatives were observed, indicating the robustness and responsiveness of the present method across different target nucleic acid copy number levels present in different clinical samples.

[0229] Example 8

[0230] Execution of this method at different temperatures

[0231] The method of the present invention can be performed efficiently over a wide range of temperatures and does not require temperature cycling, any high temperature or heating initiation, preheating, or controlled temperature reduction. This example demonstrates the performance of a general analysis over various temperature ranges. By selecting an enzyme for which the appropriate temperature is optimal and using a phosphothioate base that reduces the melting temperature of hybridization after incorporation, amplification is performed over a remarkably wide range of temperatures, and an analysis including a generally low temperature range is easily developed. Furthermore, separate experiments show that an analysis performed using the method of the present invention can be performed without the need to preheat the sample before the initiation of step a), and no loss of performance is observed when the temperature is increased during the amplification process in step a).

[0232] Example 8.1: An analysis was designed using an embodiment of the present method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a). A first primer was designed comprising a DNA target, a neutral region of 7 bases in the 5' to 3' direction; a recognition site for a restriction enzyme; and an 11-base region capable of hybridizing to a first hybridization sequence in the target nucleic acid. A second primer was designed comprising a neutral region of 7 bases in the 5' to 3' direction; a recognition site for the same restriction enzyme as the first primer; and a 12-base region capable of hybridizing to the reverse complement of a second hybridization sequence in the target nucleic acid.

[0233] A first oligonucleotide probe was designed with a total of 21 bases, comprising a 5' biotin modification in the 5' to 3' direction; a neutral region of 6 bases; a base of a restriction enzyme recognition site containing a mismatch at the second position; a 10 base region capable of hybridizing to a first hybridization region of a target containing a G-clamp modification at the sixth position; and a 3' phosphate modification, wherein the biotin modification enables the attachment of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the first oligonucleotide probe, and the phosphate modification blocks its elongation by strand-replacement DNA polymerase.

[0234] A second oligonucleotide probe was designed comprising 12 bases including an 11-base region capable of hybridizing to the reverse complement of the second hybridization sequence at the target in the 5' to 3' direction; a 4 X thymidine base spacer and a 4 X repeat of a 3-base sequence motif acting as a moiety enabling the attachment of the second oligonucleotide probe to a solid material. An additional single-stranded oligonucleotide was designed comprising an 11 X thymidine base spacer in the 5' to 3' direction; and a 33-base region including an 11 X repeat of the reverse complement to the 3-base sequence motif forming a moiety enabling the attachment of the second oligonucleotide to a solid material. For the second oligonucleotide probe, a nucleic acid lateral flow strip was prepared by spotting with 30 pmol of the additional single-stranded oligonucleotide.

[0235] A reaction was prepared comprising 1.5 pmol of the first primer; 1.0 pmol of the second primer; 1 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; and, various levels of target DNA (++ = 1 amol, + = 10 zmol, NTC = no target control). The prepared reaction was set to the target temperature (I = 37 o C; II = 45 ℃ , III = 50℃ and IV = 55 ℃ After incubating for 2 minutes, the reaction was initiated by adding 5 U of restriction enzyme and 5 U of Bacillus strand replacement DNA polymerase to a final reaction volume of 25 μl. The reaction was then incubated at the relevant target temperature for 5 minutes (T1) or 8 minutes (T2). After incubation, each reaction was transferred to 75 μl of buffer containing 1.5 pmol of the second oligonucleotide probe and 8 μg of carbon adsorbed to biotin-binding protein, and then applied to the sample pad of a nucleic acid lateral flow strip.

[0236] Figure 12a displays photographs of lateral flow strips obtained in the experiment at each target level, temperature, and time point. The observed clear black lines correspond to the deposition of carbon-attached detection species generated in the presence of the target. A very strong signal appeared within 8 minutes at both target levels in the presence of the target at all temperatures, demonstrating the wide temperature range of efficient amplification by this method. No non-specific amplification was observed in the NTC samples. Strong amplification was observed after only 5 minutes at 45°C and 50°C, indicating that the optimal temperature for this analysis is likely between 45°C and 50°C.

[0237] Example 8.2: A second analysis was designed using an embodiment of a method in which the first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by the first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a). Both the first and second primers were designed, comprising a neutral region of six bases in the 5' to 3' direction; a recognition site for the restriction enzyme; and a 12-base hybridization region for the target nucleic acid. The primers were designed such that the first and second hybridization sequences of the target are separated by 10 bases.

[0238] A first oligonucleotide probe was designed with a total length of 23 bases, comprising a 5' biotin modification in the 5' to 3' direction; a neutral region of 6 bases; a base of a restriction enzyme recognition site containing a mismatch at the 4th position; a 12-base region capable of hybridizing to a first hybridization region at the target; and a 3' phosphate modification, wherein the biotin modification enables the attachment of the first oligonucleotide probe to a colorimetric dye, carbon nanoparticles, and the first oligonucleotide probe, and the phosphate modification blocks its elongation by strand-replacement DNA polymerase.

[0239] A second oligonucleotide probe was designed comprising a 13-base region capable of hybridizing to 3 bases of the reverse complement of the second hybridization sequence in the 5' to 3' direction at the target and a 10-base gap between the first and second hybridization sequences; a 3 X thymidine base spacer and 12 bases including a 4 X repeat of a 3-base sequence motif acting as a moiety to enable attachment of the second oligonucleotide probe to a solid material. An additional single-stranded oligonucleotide was designed comprising an 11 X thymidine base spacer in the 5' to 3' direction; and a 36-base region including a 12 X repeat of the reverse complement for the 3-base sequence motif forming a moiety to enable attachment of the second oligonucleotide to a solid material. For the second oligonucleotide probe, a nucleic acid lateral flow strip was prepared by spotting 30 pmol of the additional single-stranded oligonucleotide.

[0240] A reaction was prepared containing 6 pmol of a first oligonucleotide primer; 8 pmol of a second oligonucleotide primer; 6 pmol of a first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 60 μg of carbon adsorbed to biotin-binding protein; and, where applicable, a target in an appropriate buffer. The prepared reaction was incubated for 2 minutes at the starting temperature (I = 15°C; II = 45°C), after which the reaction was initiated by adding 20 U of restriction enzyme, 20 U of Bacillus strand replacement DNA polymerase, and 40 U of reverse transcriptase to a final reaction volume of 100 μl. After the addition of the enzymes, the reaction, which started at 15°C, was immediately switched to 45°C along with other reactions.

[0241] The reaction was then incubated at 45°C for 6 minutes. After incubation, each reaction was transferred to a sample pad on a nucleic acid lateral flow strip, which contained 3 pmol of a second oligonucleotide probe. Figure 12b shows photographs of the lateral flow strips obtained in the experiment under each temperature incubation condition. The observed clear black lines correspond to the deposition of carbon-attached detection species generated 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 the amplification step a). The same significant amplification rate as in the preheating reaction occurred, and no non-specific amplification was observed in the NTC samples.

[0242] This Example 8 demonstrates that the method of the present invention can be used to facilitate analysis by having lower optimal temperature characteristics compared to known methods, and can be utilized for detection that is responsive over a very wide temperature range. It also indicates that the method of the present invention can be performed without preheating, and that the temperature increases during the performance of step a). These characteristics are very attractive when using the method in low-cost diagnostic devices, as high temperature and precise controlled heating add complex physical constraints that increase the product cost of such devices when single-use or device-free devices are not available on the market. Furthermore, by avoiding the requirement of known methods to preheat the sample before the initiation of amplification, the method of the present invention can be performed with fewer user steps and a simpler sequence of operations, thus increasing the utility of such diagnostic devices and shortening the overall time to result.

[0243] Example 9

[0244] Execution of a method in which target nucleic acids are derived from double-stranded DNA by strand penetration

[0245] This example illustrates the use of the present method for a single-stranded region within double-stranded DNA in which a single-stranded target nucleic acid is detected without the requirement for specific actions to separate the DNA strands, such as temperature denaturation, the use of bump primers, or additional enzymes (e.g., helicase or recombinase). Due to the ability to easily use the method of the present invention for the detection of both single-stranded RNA and double-stranded DNA targets, it is highly applicable for diagnostic applications without additional user steps, components, or physical requirements added to the device used to perform the method.

[0246] Example 9.1: An analysis was developed for a protein-coding region within a virus-targeted double-stranded DNA genome. It is possible to use the double-stranded genome or mRNA transcript as a biomarker for the presence of the virus in clinical diagnosis. The analysis was designed using an embodiment of the present method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and comes into contact with the sample simultaneously with the performance of step a). The design of the oligonucleotide primer and oligonucleotide probe was carried out according to an approach similar to that described in other examples, without a gap between the first and second hybridization sequences in the target nucleic acid.

[0247] In an appropriate buffer, a reaction was prepared containing 4 pmol of a first oligonucleotide primer; 2 pmol of a second oligonucleotide primer; 2 pmol of a first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 60 μg of carbon adsorbed to a biotin-binding protein; and a double-stranded DNA target (I) or a single-stranded RNA target (II), or a reaction without a target. After incubating the prepared reaction at 45°C for 2 minutes, the reaction was initiated by adding 20 U of restriction enzyme, 20 U of Bacillus strand replacement DNA polymerase, and 25 U of reverse transcriptase to a final reaction volume of 100 μl. After adding the enzymes, the reaction was incubated at 45°C for 7 minutes.

[0248] After incubation, 1.5 pmol of the second oligonucleotide probe was added to each reaction, and the entire reaction volume was transferred to the sample pads of the nucleic acid lateral flow strip. A nucleic acid lateral flow control target was also added to all samples. Figure 13a shows photographs of the lateral flow strips obtained from experiments for each target. The observed clear black lines correspond to the deposition of carbon-attached detection species generated in the presence of the target, and faint signals correspond to lower target levels (+) than higher target levels (++). No difference in amplification rates was observed between single-stranded RNA and double-stranded DNA targets.

[0249] Example 9.2: An analysis was designed for a single-stranded target nucleic acid within the c.2.5 megabase double-stranded DNA genome of a bacterial pathogen. The analysis was designed using an embodiment of the present method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and comes into contact with the sample simultaneously with the performance of step a). The design of the oligonucleotide primer and oligonucleotide probe was carried out according to an approach similar to that described in another example, in which a four-base gap exists between the first and second hybridization sequences in the target nucleic acid.

[0250] A reaction was prepared containing 2 pmol of a first oligonucleotide primer and 0.5 pmol of a second oligonucleotide primer in an appropriate buffer. It is assumed that due to the use of a double-stranded DNA target, two single-stranded target nucleic acids are actually added simultaneously and a second interaction process is also triggered, and the second oligonucleotide primer for target nucleic acid detection is the first oligonucleotide primer for the detection of the second target nucleic acid, which is the anti-complement of the target nucleic acid. This fact has little effect on the performance of the present method. 2 pmol of the first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM each of dTTP, dCTP, and dGTP; 15 μg of carbon adsorbed to biotin-binding proteins; and bacterial genome extracts containing various levels of target (++ = 1 amol; + = 10 zmol; NTC = no target control). An additional specificity control reaction containing 1 amol of E. coli genome extract was also performed. After incubating the prepared reaction at 45°C for 3 minutes, 4 U of restriction enzyme and 2 U of Bacillus strand replacement DNA polymerase were added at the end to a final reaction volume of 25 μl to initiate the reaction. After adding the enzymes, the reaction was incubated at 45°C for 6 minutes.

[0251] After incubation, 75 μl of buffer containing 3 pmol of the second oligonucleotide probe was added to each reaction, and the entire volume was transferred to the sample pad of the nucleic acid lateral flow strip. Figure 13b shows photographs of the lateral flow strips obtained from experiments for each target. A clear black line corresponding to the deposition of carbon-attached detection species generated in the presence of the target is observed at the levels of the two tested targets. No non-specific signals were observed in the presence of the targetless control or E. coli genomic DNA, indicating that the present method can be used for the specific detection of complex double-stranded DNA genomes at clinically relevant copy numbers of only six or fewer.

[0252] This example demonstrates that the method of the present invention can be readily used for the detection of single-stranded nucleic acid targets within double-stranded DNA. Surprisingly, similar amplification rates are observed for the detection of single-stranded RNA targets and the same target sequences within double-stranded DNA without the requirement for specific actions, such as temperature denaturation, to separate DNA duplexes. Instead, the method is initiated by "strand penetration" by sufficiently exposing the single-stranded region to hybridization and extension of the first oligonucleotide primer, thereby allowing for sufficient transient opening of one or more DNA base pairs within the double-stranded DNA to enable hybridization and extension of the 3' hydroxyl of the first oligonucleotide primer. This differs from known methods, such as SDA, where heat denaturation and bump primers are used in the analysis of double-stranded nucleic acids. Due to the ability to readily detect targets within double-stranded DNA in addition to targets within single-stranded DNA and single-stranded RNA using the method of the present invention, it can be widely used in diagnostic applications, such as the detection of bacterial, fungal, and viral pathogens with double-stranded DNA genomes. The fact that this method requires no complex additional user steps, enzymes, components, or physical constraints to detect organisms with double-stranded DNA genomes means that it is particularly suitable for testing on simple and low-cost diagnostic devices. For example, the requirement for thermal denaturation prior to amplification reported for known methods requires expensive additional components and increases the product cost and total time to result for such devices, which means that single-use or self-contained, instrument-free devices are not feasible.

[0253] Example 10

[0254] Performing a comparison between the method of the present invention and a known method

[0255] This example provides a comparative evaluation of the method of the present invention with the known method disclosed in WO2014 / 164479 for the detection of a viral target. The known method differs fundamentally from the method of the present invention in that it requires a nick-forming enzyme and does not require the use of one or more modified dNTPs. The method of the present invention has been proven to have excellent responsiveness and specificity.

[0256] For this comparative evaluation, an analysis of a viral target having a single-stranded RNA genome was first performed using the method of the present invention. The analysis was designed using an embodiment of the present method in which a first oligonucleotide probe is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by a first or second restriction enzyme, and comes into contact with the sample simultaneously with the performance of step a). The design of the oligonucleotide primer and oligonucleotide probe was carried out according to an approach similar to that described in other examples, with a six-base gap between the first and second hybridization sequences in the target nucleic acid.

[0257] For analysis using the disclosed method, similar primers were designed as equivalent primers used in the method of the present invention, comprising the same six-base neutral region at the 5' end and the same hybridization region at the 3' end. In this way, maximum consistency between the two analyses was ensured for an accurate comparison of the methods. However, the bases of the restriction enzyme recognition site were replaced with the bases of the exemplary nick-forming enzyme reported in WO2014 / 164479, Nt.BbvCI (see Example 5 on pages 20 to 21).

[0258] Example 10.1: In the first case, the reaction for each method was performed using the same primer ratio. For the method of the present invention, a reaction was prepared comprising 2 pmol of a first oligonucleotide primer; 2 pmol of a second oligonucleotide primer; 1.6 pmol of a first oligonucleotide probe; 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM of each of dTTP, dCTP, and dGTP; and various levels of viral genomic RNA as targets (+++ = 10 zmol; ++ = 100 copies; + = 10 copies; NTC = untargeted control) in a suitable buffer. After pre-incubating the prepared reaction for 5 minutes under ambient conditions (c. 20°C), the reaction was initiated by adding 5 U of restriction enzyme, 5 U of Bacillus strand replacement DNA polymerase, and 10 U of reverse transcriptase to a final reaction volume of 25 μl. After adding the enzyme, the reaction was incubated at 45°C for 8 minutes (T1) or 15 minutes (T2). After incubation, 60 μg of carbon adsorbed to biotin-binding proteins in 75 μl of buffer was added to each reaction, and the total volume of 100 μl was transferred to a sample pad with a nucleic acid lateral flow strip containing 1.5 pmol of a second oligonucleotide probe.

[0259] For the known method, a reaction was prepared containing 6.25 pmol of a first oligonucleotide primer; 6.25 pmol of a second oligonucleotide primer; 200 μM of each of dATP, dTTP, dCTP, and dGTP; and various levels of viral genomic RNA as targets (+++ = 10 zmol; ++ = 100 copies; + = 10 copies; NTC = no target control). After pre-incubating the prepared reaction at ambient conditions (c. 20°C) for 5 minutes, the reaction was initiated by adding 4 U of Nt.BbvCl, 20 U of Bst large fragment DNA polymerase, and 10 U of M-MuLV reverse transcriptase to a final reaction volume of 25 μl. After enzyme addition, the reaction was incubated at 45°C for 8 minutes (T1) or 15 minutes (T2). After incubation, 60 μg of carbon adsorbed to biotin-binding protein and 5 pmol of the first oligonucleotide probe in 75 μl of buffer were added to each reaction, and the total volume of 100 μl was transferred to a sample pad with a nucleic acid lateral flow strip containing 5 pmol of the second oligonucleotide probe.

[0260] FIG. 14a shows photographs of lateral flow strips obtained in experiments using the method (I) of the present invention and the known method (II) at various indicated target levels and time points. The observed black lines correspond to the deposition of carbon-attached detection species generated in the presence of the target. Using the known method, multiple attempts were required to observe any signal, and specific combinations of enzymes and buffers, as well as significantly higher levels of primers, dNTPs, and enzymes, had to be used. Using the method of the present invention (I), detection species generated could be clearly observed at the lowest target level of only 10 target copies, even at the shortest time point, after only 8 minutes without preheating. Even after efforts to optimize the known method, which was not obvious to those skilled in the art, only faint signals were observed at the highest target level (+++ = 10 zmol) and the longest time point (15 minutes).

[0261] Example 10.2: After more extensive and less obvious attempts, it was possible to increase the performance of the known method, but only by using a very high concentration of the first primer and a 2:1 ratio of first and second oligonucleotide primers as described in Example 10.2. The method of the present invention was performed again as described in Example 10.1. For the known method, the reaction was performed as described in Example 10.1, except that the level of the first oligonucleotide primer was increased to 12.5 pmol. In each case, the following target levels were used: +++ = 1 zmol; ++ = 100 copies; + = 10 copies; NTC = no target control.

[0262] FIG. 14b shows photographs of lateral flow strips obtained in experiments using the method (I) of the present invention and the known method (II) at various indicated target levels and time points. The observed black lines correspond to the deposition of carbon-attached detection species generated in the presence of the target. Again, the method (I) of the present invention demonstrated a remarkable rate in which a signal was observable even at the shortest time point and the lowest target level of only 10 target copies. Using the known method, only a faint signal was observed at the highest target level (+++ = 1 zmol), and a very faint signal was observed in 100 copy samples at the longest time point (15 min). However, a faint signal was also observed in the NTC strip, which may correspond to a non-specific product resulting from very high oligonucleotide primer levels and enzyme levels required for the method to operate. These data are consistent with the data from WO2014 / 164479, which reported an incubation time of 30 min. The requirement to add abnormally high primer levels to increase the amplification rate performed using this disclosed method will significantly limit the applicability to the detection of two or more different targets in the same sample, as the range for further increase to further increase the total primer level without exacerbating the problem of non-specific products is very limited.

[0263] Example 14 demonstrates the remarkable superiority of the method of the present invention over the known method disclosed in WO2014 / 164479, in that amplification is performed at a much faster speed and generates greater responsiveness and a clearer resulting signal. In just 8 minutes without pre-incubation, the method of the present invention generated a stronger signal with only 100 target copies than the known method was able to achieve within 15 minutes at the highest target level of 60X. The advantage of the method of the present invention over the known method arises from the requirement for a different type of enzyme, such as a restriction enzyme rather than a nick-forming enzyme, and the requirement for the use of one or more modified dNTPs, such as phosphothioate bases, which improves the responsiveness and specificity of the amplification. Furthermore, an embodiment of the method in which one of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end, is not cleaved by the first or second restriction enzyme, and contacts the sample simultaneously with the performance of step a), enables efficient linkage of amplification for signal detection and facilitates enhanced specificity derived from efficient sequence-based hybridization during the formation of the detection species. Due to these advantages, the method of the present invention is ideally suited for applications in the diagnostic field and for the development of simple, ultra-fast, user-centric, low-cost diagnostic devices, such as single-use or instrument-free molecular diagnostic test devices. Unless the context otherwise requires, throughout the specification and appended claims, the word “comprising” and variations such as “comprising” and “comprising” will be understood to mean that it includes the specified integer, step, group of integers, or group of steps, but does not exclude any other integer, step, group of integers, or group of steps.

[0264] Further aspects of the present invention include those listed below:

[0265] 1. A method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample,

[0266] a) to generate an amplification product without temperature cycling in the presence of the above target nucleic acid

[0267] i. A first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid;

[0268] ii. strand replacement DNA polymerase;

[0269] iii. dNTP;

[0270] iv. One or more modified dNTPs;

[0271] v. A first restriction enzyme that is not a nick-forming enzyme, but is capable of recognizing the recognition sequence of a first primer and cleaving only the first primer strand at the cleaving site when the recognition sequence and the cleaving site are double-stranded, wherein the cleaving of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a DNA polymerase using one or more modified dNTPs; and

[0272] vi. A second restriction enzyme that is not a nick-forming enzyme, but where the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and cleave only the second primer strand at the cleavage site, wherein the cleavage of the anti-complement strand is blocked by the presence of one or more modifications incorporated into the anti-complement strand by a DNA polymerase using one or more modified dNTPs.

[0273] Step of contacting the sample;

[0274] b)

[0275] i. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and

[0276] ii. A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence among the amplification products of at least one species, and attached to a solid material or a moiety that enables attachment to a solid material.

[0277] as a step of contacting the amplification product of step a);

[0278] Hybridization of the first and second probes for at least one species in the amplification product to generate a detection species; and

[0279] c) a step of detecting the presence of a detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample

[0280] A method including

[0281] 2. A method according to aspect 1, wherein one of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme.

[0282] 3. A method in which, in aspect 2, one oligonucleotide probe is not cleaved by a first or second restriction enzyme due to the presence of one or more sequence mismatches and / or one or more modifications, such as phosphorothioate linkages.

[0283] 4. A method in which, in aspect 2 or 3, one oligonucleotide probe is in contact with the sample simultaneously with the performance of step a).

[0284] 5. In any one of aspects 1 to 4, the sample is further contacted in step a) (A) a third oligonucleotide primer comprising a recognition sequence and a cleavage site of a first restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, said third oligonucleotide primer which is blocked from elongation by DNA polymerase at the 3' end; and / or (B) a fourth oligonucleotide primer comprising a recognition sequence and a cleavage site of a second restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to the reverse complement of a second hybridization sequence in the target sequence, said fourth oligonucleotide primer which is blocked from elongation by DNA polymerase at the 3' end.

[0285] 6. A method according to aspect 5, wherein, provided, the third oligonucleotide primer is provided in excess of the first oligonucleotide primer, and, where provided, the fourth oligonucleotide primer is provided in excess of the second oligonucleotide primer.

[0286] 7. A method in which, in any one of aspects 1 to 6, one or more modified dNTPs are alpha-thiol modified dNTPs.

[0287] 8. A method in which, in any one of aspects 1 to 7, the first and second restriction enzymes are the same restriction enzyme.

[0288] 9. A method in which, in any one of aspects 1 to 8, two or more of steps a), b), and c) are performed simultaneously.

[0289] 10. In any one of aspects 1 to 9, step (a) is performed at a temperature of 50°C or lower.

[0290] 11. A method in any one of aspects 1 to 10, wherein the moiety enabling detection of the first oligonucleotide probe is a moiety capable of attaching to a colorimetric or fluorescent dye or a colorimetric or fluorescent dye such as biotin.

[0291] 12. A method in which, in any one of aspects 1 to 11, the detection species is detected by a change in an electrical signal.

[0292] 13. A method in any one of aspects 1 to 12, wherein the moiety enabling detection of the first oligonucleotide probe is an enzyme that generates a detectable signal, such as a colorimetric or fluorescent signal, after contact with a substrate.

[0293] 14. A method in any one of aspects 1 to 13, wherein the moiety enabling attachment of the second oligonucleotide probe to the solid material is a single-stranded oligonucleotide.

[0294] 15. A method according to aspect 14, wherein the sequence of the single-stranded oligonucleotide moiety comprises three or more repeat copies of a 2 to 4 base DNA sequence motif.

[0295] 16. A method in which, in any one of aspects 1 to 15, the presence of a detected species in step c) is detected by nucleic acid lateral flow.

[0296] 17. A method according to aspect 16, wherein the nucleic acid lateral flow enables sequence-specific hybridization of a moiety that enables attachment of a second oligonucleotide probe to a solid material.

[0297] 18. In any one of aspects 1 to 17, step c) is a method of generating a colorimetric or electrochemical signal using carbon or gold, preferably carbon.

[0298] 19. A method in any one of aspects 1 to 18, wherein the first and / or second oligonucleotide primer comprises a restriction enzyme recognition sequence and a stabilization sequence of, for example, 5 base lengths at the 5' end upstream of the cleavage site.

[0299] 20. A method in any one of aspects 1 to 19, wherein the hybridization region of the first and / or second oligonucleotide primer is 9 to 16 bases long.

[0300] 21. A method in any one of aspects 1 to 20, wherein one of the first and second oligonucleotide primers is provided in excess of the other.

[0301] 22. A method in which, in any one of aspects 1 to 21, the first and second hybridization sequences in the target nucleic acid are separated by 0 to 6 bases.

[0302] 23. A method in which, in any one of aspects 1 to 22, the first and second hybridization sequences in the target nucleic acid are separated by 3 to 6 bases.

[0303] 24. A method in which, in any one of aspects 1 to 23, the first or second single-strand detection sequence of at least one species in the amplification product in step b) comprises a sequence corresponding to 3 to 6 bases as defined in claim 23.

[0304] 25. A method in which, in any one of aspects 1 to 24, the level of the target nucleic acid in the sample is quantified in step c).

[0305] 26. A method in any one of aspects 1 to 25, wherein the target nucleic acid is a single-stranded RNA comprising a single-stranded RNA derived from double-stranded RNA and a single-stranded RNA derived from double-stranded DNA, or a single-stranded DNA comprising a single-stranded DNA derived from single-stranded RNA and a single-stranded DNA derived from double-stranded DNA.

[0306] 27. A method according to aspect 26, wherein the single-stranded DNA is derived from double-stranded DNA by the use of a nuclease, e.g., restriction endonuclease or exonuclease III, or derived from single-stranded RNA by the use of a reverse transcriptase.

[0307] 28. A method in which, in any one of aspects 1 to 27, the presence of two or more different target nucleic acids of a predetermined sequence is detected in the same sample.

[0308] 29. A method in which, in any one of aspects 1 to 28, the sample is a biological sample such as a nasal or nasopharyngeal swab or adsorbate, blood, or a sample derived from blood or urine.

[0309] 30. A method in any one of aspects 1 to 29, wherein the target nucleic acid is a virus, derived from viral nucleic acid material, a bacterium, derived from bacterial nucleic acid material, circulating cell-free DNA released from a cancer cell or fetal cell, a microRNA, or derived from microRNA.

[0310] 31. A method in any one of aspects 1 to 30, wherein the target nucleic acid comprises an epigenetic modification site such as methylation.

[0311] 32. A method in any one of aspects 1 to 31, wherein the detection of the target nucleic acid is used for the diagnosis, prognosis, or monitoring of a disease or disease state.

[0312] 33. A method according to aspect 32, wherein the 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, bubonic plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox.

[0313] 34. Aspect 32, wherein the 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.

[0314] 35. A method in any one of aspects 1 to 34, wherein the detection of the target nucleic acid is used in human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics.

[0315] 36. A method in any one of aspects 1 to 35, wherein the sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biological defense sample.

[0316] 37.

[0317] a) a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence, a cleavage site, and a first hybridization sequence within a single-stranded target nucleic acid of a predetermined sequence in the 5' to 3' direction, and the second primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence, a cleavage site, and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence within the target nucleic acid in the 5' to 3' direction;

[0318] b) a first restriction enzyme that is not a nick-forming enzyme but can cleave a cleavage site by recognizing the recognition sequence of a first primer, and a second restriction enzyme that is not a nick-forming enzyme but can cleave a cleavage site by recognizing the recognition sequence of a second primer;

[0319] c) strand replacement DNA polymerase;

[0320] d) dNTP;

[0321] e) One or more modified dNTPs;

[0322] f) a first oligonucleotide probe that is partially complementary to the hybridization region of one of the first and second oligonucleotide primers and is attached to a moiety that enables detection; and

[0323] g) A second oligonucleotide probe that is partially complementary to the inverse complement of the hybridization region of the other of the first and second oligonucleotide primers and attaches to a solid material or a moiety that enables attachment to a solid material.

[0324] A kit including

[0325] 38. A kit according to aspect 37, further comprising means for detecting the presence of a detection species.

[0326] 39. In aspect 37 or 38, 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 dNTP and / or one or more modified dNTPs and / or the first oligonucleotide probe and / or the second oligonucleotide probe is a kit as defined in any one of aspects 2, 3, 7, 8, 11, 13 to 17, 19, 20 or 22 to 24.

[0327] 40. A kit comprising, in any one of aspects 37 to 39, a third and / or fourth oligonucleotide primer as defined in aspect 5 or 6.

[0328] 41. A method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample, wherein

[0329] a) to generate an amplification product without temperature cycling in the presence of the above target nucleic acid

[0330] i. A first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid;

[0331] ii. strand replacement DNA polymerase;

[0332] iii. dNTP;

[0333] iv. One or more modified dNTPs;

[0334] v. A first restriction enzyme that is not a nick-forming enzyme, but is capable of recognizing the recognition sequence of a first primer and cleaving only the first primer strand at the cleaving site when the recognition sequence and the cleaving site are double-stranded, wherein the cleaving of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into the reverse complement strand by a DNA polymerase using one or more modified dNTPs; and

[0335] vi. A second restriction enzyme that is not a nick-forming enzyme, but where the recognition sequence and the cleavage site are double-stranded, can recognize the recognition sequence of the second primer and cleave only the second primer strand at the cleavage site, wherein the cleavage of the anti-complement strand is blocked by the presence of one or more modifications incorporated into the anti-complement strand by a DNA polymerase using one or more modified dNTPs.

[0336] Step of contacting the sample;

[0337] b)

[0338] i. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and

[0339] ii. A second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence among the amplification products of at least one species, and attached to a solid material or a moiety that enables attachment to a solid material.

[0340] as a step of contacting the amplification product of step a);

[0341] One of the first and second oligonucleotide probes is blocked from elongation by DNA polymerase at the 3' end and is not cleaved by the first or second restriction enzyme, and hybridization of the first and second probes for at least one species in the amplification product generates a detection species; and

[0342] c) a step of detecting the presence of a detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample,

[0343] The first or second oligonucleotide probe specified in step b) is in contact with the sample simultaneously with the execution of step a).

[0344] A method including

[0345] All patents and patent applications mentioned in this specification are incorporated by reference in their entirety.

Claims

Claim 1 A method for detecting the presence of a single-stranded target nucleic acid of a predetermined sequence in a sample, comprising: a) i. a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and a first hybridization sequence in the target nucleic acid, and the second primer comprises a region capable of hybridizing to a single-stranded restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction and an inverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid; ii. a strand-alternating DNA polymerase; iii. a dNTP; iv. one or more modified dNTPs; v. A first restriction enzyme, wherein the first restriction enzyme is not a nick-forming enzyme, but when the recognition sequence and cleavage site of the first primer are double-stranded, the first restriction enzyme can recognize said recognition sequence and can cleave only the first primer strand at the cleavage site, and the cleavage of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into said reverse complement strand by a DNA polymerase using one or more modified dNTPs; and vi. a step of contacting a sample with a second restriction enzyme, wherein the second restriction enzyme is not a nick-forming enzyme, but when the recognition sequence and cleavage site of the second primer are double-stranded, the second restriction enzyme can recognize said recognition sequence and can cleave only the second primer strand at the cleavage site, and the cleavage of the reverse complement strand is blocked due to the presence of one or more modifications incorporated into said reverse complement strand by a DNA polymerase using one or more modified dNTPs; b) i. the amplification product of step a). ii. A first oligonucleotide probe capable of hybridizing to a first single-stranded detection sequence of at least one species in the amplification product and attached to a moiety that enables the detection thereof; and ii.A method comprising: contacting a second oligonucleotide probe that is capable of hybridizing to a second single-strand detection sequence upstream or downstream of a first single-strand detection sequence of the same strand of at least one species in the amplification product and is attached to a moiety that enables attachment to or to a solid material; at least one of the first and second oligonucleotide probes is cut off at the 3' end from extension by DNA polymerase and is not cleaved by a first or second restriction enzyme; the cut-off oligonucleotide probe is contacted with a sample simultaneously with the performance of step a), and hybridization of the first and second probes for at least one species in the amplification product generates a detection species; and detecting the presence of the detection species generated in step b), wherein the presence of the detection species indicates the presence of a target nucleic acid in the sample. Claim 2 The method of claim 1, wherein at least one blocked oligonucleotide probe is not cleaved by a first or second restriction enzyme due to the presence of one or more sequence mismatches or one or more modifications, said one or more modifications being phosphorothioate linkages. Claim 3 The method of claim 1, wherein at least one blocked oligonucleotide probe comprises an additional region that allows the 3' end of a species in the amplification product hybridized by the blocked oligonucleotide probe to be extended by a strand replacement DNA polymerase. Claim 4 In claim 1, the sample is further contacted in step a) (A) as a third oligonucleotide primer, wherein the third primer comprises a single strand recognition sequence and a cleavage site for a first restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to a first hybridization sequence in the target nucleic acid, said third primer is cut off at the 3' end from extension by DNA polymerase; and (B) as a fourth oligonucleotide primer, said fourth primer comprises a single strand recognition sequence and a cleavage site for a second restriction enzyme in the 5' to 3' direction and a region capable of hybridizing to the anti-complement of a second hybridization sequence in the target nucleic acid, said fourth primer is cut off at the 3' end from extension by DNA polymerase; where present, the third oligonucleotide primer is provided in excess of the first oligonucleotide primer; A method in which, if present, the fourth oligonucleotide primer is provided in excess of the second oligonucleotide primer. Claim 5 The method of claim 1, wherein one or more modified dNTPs are alpha-thiol modified dNTPs. Claim 6 A method according to claim 1, wherein the first and second restriction enzymes are the same restriction enzyme. Claim 7 A method according to claim 1 or 2, wherein step a) is performed at a temperature of 50°C or lower, or the temperature is increased during the performance of step a), wherein the increase from the ambient starting temperature is from a range of 15 to 30°C up to a temperature within a range of 40 to 50°C. Claim 8 A method according to claim 1, wherein the moiety enabling detection of the first oligonucleotide probe is a colorimetric or fluorescent dye, or a moiety capable of attaching to a colorimetric or fluorescent dye, which is a biotin moiety, or an enzyme that generates a detectable signal after contact with a substrate, wherein the signal is a colorimetric or fluorescent signal. Claim 9 A method according to claim 1 or 2, wherein the detection species is detected by a change in an electrical signal. Claim 10 A method according to claim 1, wherein the moiety enabling attachment of the second oligonucleotide probe to a solid material is a single-stranded oligonucleotide, and the sequence of the single-stranded oligonucleotide moiety may include three or more repeat copies of a DNA sequence motif of 2 to 4 bases. Claim 11 A method according to claim 1 or 2, wherein the presence of a detected species is detected by a nucleic acid lateral flow, and said lateral flow is a nucleic acid lateral flow capable of sequence-specific hybridization of a moiety that enables attachment of a second oligonucleotide probe to a solid material. Claim 12 A method according to claim 1 or 2, wherein the detecting step comprises the step of generating a colorimetric or electrochemical signal using carbon or gold. Claim 13 A method according to claim 1, wherein the first and second oligonucleotide primers comprise a restriction enzyme recognition sequence and a stabilization sequence upstream of the cleavage site. Claim 14 The method of claim 1, wherein the hybridization regions of the first and second oligonucleotide primers are 9 to 16 bases long. Claim 15 A method according to claim 1, wherein one of the first and second oligonucleotide primers is provided in excess of the other. Claim 16 A method according to claim 1, wherein the first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 bases. Claim 17 A method according to claim 1, wherein the first and second hybridization sequences in the target nucleic acid are separated by 3 to 15 bases, and in step b), the first or second single-stranded detection sequence of at least one species in the amplification product comprises at least 3 bases of a sequence corresponding to the 3 to 15 bases. Claim 18 A method according to claim 1, wherein the target nucleic acid is a single-stranded RNA comprising a single-stranded RNA derived from double-stranded RNA and a single-stranded RNA derived from double-stranded DNA, or a single-stranded DNA comprising a single-stranded DNA derived from single-stranded RNA and a single-stranded DNA derived from double-stranded DNA, wherein the single-stranded DNA is derived from double-stranded DNA by strand penetration or derived from double-stranded DNA by the use of a nuclease. Claim 19 A method according to claim 1, wherein the presence of two or more different target nucleic acids of a predetermined sequence is detected in the same sample. Claim 20 In paragraph 1, the sample is a biological sample, a biological sample that is a nasal or nasopharyngeal swab or adsorbate, blood or a sample derived from blood, or urine; a human sample; a forensic sample; an agricultural sample; a veterinary sample; an environmental sample; or a biodefense sample. Claim 21 A method according to claim 1, wherein the target nucleic acid is a virus or derived from viral nucleic acid material, a bacterium or derived from bacterial nucleic acid material, circulating cell-free DNA released from cancer cells or fetal cells, or microRNA or derived from microRNA. Claim 22 In paragraph 1, the detection of the target nucleic acid is used to provide information regarding the diagnosis, prognosis, or monitoring of a disease or disease state, wherein the disease or disease state 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, bubonic plague, polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox, or a colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, kidney cancer, gastric cancer, or melanoma, A method in which the detection of target nucleic acids is used to provide information regarding human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics, or pharmacokinetics. Claim 23 A kit for use in detecting the presence of a single-stranded target nucleic acid in a sample using the method of claim 1, comprising: a) a first oligonucleotide primer and a second oligonucleotide primer, wherein the first primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence and a cleavage site and a first hybridization sequence in a single-stranded target nucleic acid of a predetermined sequence in the 5' to 3' direction, and the second primer comprises a region capable of hybridizing to a restriction enzyme recognition sequence and a cleavage site and a reverse complement of a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid in the 5' to 3' direction; b) a first restriction enzyme that is not a nick-forming enzyme but recognizes the recognition sequence of the first primer and cleaves the cleavage site, and a second restriction enzyme that is not a nick-forming enzyme but recognizes the recognition sequence of the second primer and cleaves the cleavage site; c) a strand-alternating DNA polymerase; d) a dNTP; e) one or more modified dNTPs; f) a first oligonucleotide probe capable of hybridizing to a first single-strand detection sequence of at least one species in an amplification product generated in the presence of the target nucleic acid and attached to a moiety that enables such detection; and g) a second oligonucleotide probe capable of hybridizing to a second single-strand detection sequence upstream or downstream of the first single-strand detection sequence of the same strand of at least one species in the amplification product and attached to a moiety that enables attachment to or to a solid material, wherein at least one of the first and second oligonucleotide probes is cut off at the 3' end from extension by the DNA polymerase and is not cleaved by the first or second restriction enzyme. Claim 24 In claim 23, at least one blocked oligonucleotide probe is blocked at the 3' end from extension by DNA polymerase and is not cleaved by a first or second restriction enzyme due to the presence of one or more sequence mismatches or one or more modifications, said one or more modifications being phosphorothioate linkages, Kit. Claim 25 A kit according to claim 23 or 24, wherein one of the first and second oligonucleotide probes has five or more bases that are complementary to the hybridization region or the reverse complement of the hybridization region of the first or second primer. Claim 26 A kit according to claim 24, wherein the first oligonucleotide probe has five or more bases complementary to the hybridization region of one of the first and second oligonucleotide primers, and the second oligonucleotide probe has five or more bases complementary to the inverse complement of the hybridization region of the other of the first and second oligonucleotide primers. Claim 27 A kit according to claim 23 or 24, further comprising means for detecting the presence of a detection species generated in the presence of a target nucleic acid. Claim 28 In paragraph 27, the means for detecting the presence of a detection species is a kit, which is a colorimetric or electrochemical signal using carbon or gold. Claim 29 In claim 23 or 24, the target nucleic acid, the first oligonucleotide primer, the second oligonucleotide primer, the first restriction enzyme, the second restriction enzyme, the DNA polymerase, the dNTP, one or more modified dNTPs, the first oligonucleotide probe, the second oligonucleotide probe, and the first or second single-stranded detection sequence of at least one species in the amplification product are as specified in any one of claims 3, 5, 6, 8, 10, and 13 to 22, kit. Claim 30 A kit according to claim 23 or 24, further comprising third and fourth oligonucleotide primers as prescribed in claim 4. Claim 31 A device comprising a kit according to paragraph 23 or 24, wherein the device comprises a power supply unit; a heating means; or a single-use diagnostic device. Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete

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