Nucleic acid detection method

Abstract

To provide methods for the detection of nucleic acids of defined sequence.SOLUTION: The present invention provides methods that employ restriction enzymes, polymerase, and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, where the amplification product is brought into contact with oligonucleotide probes to produce a detector product.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] (Related Technology) Polymerase-based nucleic acid sequence amplification methods are widely used in the field of molecular diagnostics. The polymerase chain reaction (PCR), the most established method, typically uses two PCR primers for each target sequence. It involves two primers and uses periodic changes in temperature in a process of cyclic exponential amplification. The primers are annealed, extended by DNA polymerase, and then newly synthesized The requirement for temperature cycling requires complex equipment, which is This limits the use of PCR-based methods in clinical applications.

[0003] Strand Displacement Amplification (SDA) (EP0497272; US5455166; US5712124) was developed as an alternative to isothermal PCR, which amplifies double-stranded DNA during polymerase amplification. No temperature cycling is required to achieve annealing and denaturation of A, but instead strand displacement. A restriction enzyme combined with a PCR-type polymerase is used to separate the two DNA strands.

[0004] In SDA, the restriction enzyme site at the 5' end of each primer is converted to one or more α-thiol nucleoside fragments. In the presence of a restriction enzyme, the unmodified hemiphosphate ions are introduced into the amplification product, and the restriction enzyme is used to amplify the unmodified hemiphosphate ions at its recognition site. Nicks the restriction site due to its ability to cleave only the strand in phosphorothioate form Strand-displacing polymerases extend the 3' end of each nick, displacing the downstream DNA strand. Exponential amplification results in the pairing of sense and antisense reactants. The strand displaced from the nucleotide serves as the target for the antisense reactant, and vice versa. SDA typically takes more than an hour to perform, which limits its utility in the field of clinical diagnostics. Moreover, the specific detection of the amplified products and the initiation of the reaction are significantly limited. This approach adds significant complexity due to the requirement of separate processes for

[0005] Subsequently, Maples et al. (WO2009 / 012246) reported that the nucleotide sequence bound to the specific double-stranded recognition sequence Nitroso is a subclass of restriction enzymes that can cut only one of the two strands of DNA after They performed SDA using a nicking enzyme. They described this method as a nicking and extension amplification reaction (N NEAR, which uses nicking enzymes rather than restriction enzymes, was also developed later. Using primers optimized by the software (WO2014 / 164479), and warm start Other researchers have attempted to improve this method through controlled temperature reduction (WO2018 / 002649). However, only a few nicking enzymes are available. Therefore, it is more difficult to find an enzyme with the desired properties for a particular application.

[0006] A very serious drawback of SDA (NEAR) using restriction enzymes or nicking enzymes is that this method The method produces double-stranded nucleic acid products, thus providing an essential process for efficient detection of the amplified signal. This limits its usefulness in, for example, low-cost diagnostic devices. The double-stranded nature of the amplification products produced limits the amplification method to signal detection. This presents a challenge for hybridization without first separating the two strands. This is because it is not possible to perform reduction-based detection. Coarse detection methods, such as molecular beacons and fluorophore / quencher probes, are required. This can complicate the assay protocol by requiring separate process steps. This significantly reduces the potential for developing multiplex assays.

[0007] To overcome the limitations of SDA, a rapid, highly sensitive and specific method for detecting nucleic acid sequences is required. The present invention provides an enhanced amplification method for the detection of nucleotides by using a primer pair having a 5' restriction site. In addition, an additional oligonucleotide probe is utilized to allow efficient signal detection. The present invention relates to a method for amplifying and detecting a target nucleic acid sequence, which produces a detector species. Summary of the Invention

[0008] (overview) The present invention is a method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample. Is this: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; and inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) Purifying the amplification product of step a): i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is conjugated to a moiety that enables ligation; contacting said first and second amplicons with said at least one species in said amplification product; Hybridization of the lobes produces a detector species. and c) detecting the presence of the detector species produced in step b), the presence of a target species indicates the presence of the target nucleic acid in the sample. The method further comprises:

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

[0010] In various embodiments, the methods are ideally suited for sensitive detection of the presence of target nucleic acid. The detector rapidly produces many copies of the desired detector species.

[0011] The present invention in various aspects provides an essential process for efficient detection of amplification products. In addition, it is more effective than known techniques because it involves rapid amplification without temperature cycling. It is beneficial.

[0012] The method of the present invention allows SDA to efficiently detect the amplification signal due to the double-stranded nature of the amplified product. SDA(NEAR) containing nicking enzyme does not provide an essential process for This method overcomes the major drawback of DA. The method hybridizes with at least one species in the amplification product. Two additional oligonucleotide probes were used to size the IgG1A antibody to facilitate its rapid and specific detection. This limitation is overcome by utilizing these two additional oligonucleotide probes. The use of lobes provides the present invention with several additional advantages over known methods such as SDA. The first oligonucleotide probe is bound to a moiety that allows its detection, and the second oligonucleotide probe is bound to a moiety that allows its detection. The oligonucleotide probe is bound to a solid material or to a moiety that allows for binding to the solid material. For example, one of the oligonucleotide probes can be used to bind to the DNA polymerase. The extension by the restriction enzyme is blocked at the 3' end, and the restriction enzyme cannot cut the fragment. In embodiments of the invention in which the sample is contacted with a nucleic acid molecule, unexpectedly no significant adverse inhibition of amplification is observed. This is not expected, and pre-detector species containing single-stranded regions are efficiently produced. In an embodiment, such blocked probes are used to detect counter amplification products included in the predictor species. This is counterintuitive since it may be assumed that this leads to asymmetric amplification that is biased towards one strand. The detector species is efficiently produced and ideally suited for efficient detection. The double-stranded region is readily available for hybridization with other oligonucleotide probes This is because.

[0013] The inherent sample detection approach of the method involves, for example, detecting one amplicon strand more than the other. By using unequal primer ratios to produce excess Previous attempts to overcome the significant limitations of SDA, including implementing "amplification," have been fundamentally In contrast, the present method does not require asymmetric amplification and does not selectively amplify one strand of the amplicon more than the other. Instead, the first and second oligonucleotide probes need not be produced in large quantities in excess. The focus is on the production of detector species after hybridization. The essential sample detection approach of the present method, which includes, ideally, other detection methods, particularly step c ), providing a simplified, rapid, and low-cost means of performing detection in, for example, a second By printing oligonucleotide probes of This method is suitable for combination with nucleic acid lateral flow. Together, they also each have a different sequence designed for a different target nucleic acid sequence in the sample. A plurality of second oligonucleotides are attached to the lateral flow strip at discrete locations. This allows for an efficient multiplex system based on differential hybridization of tide probes. In a further embodiment of the method, the The use of single-stranded oligonucleotides as moieties that allow for binding to solid materials allows for labeling. The efficiency of transverse flow detection is enhanced by printing the reverse complementary sequence of the moiety on the strip. The latter approach also allows for the creation of a single “universal” assay across multiple target applications. This allows the lateral flow strip to be optimized and manufactured as a delivery system. The sequence bound to the teral flow strip can be defined, and the target nucleic acid Therefore, the sequence of the 2 The essential requirement of two additional oligonucleotide probes is the SDA with nicking enzyme. It offers many advantages over SDA, including (NEAR).

[0014] The present invention requires the use of a restriction enzyme that is not a nicking enzyme and one or more modified dNTPs. Therefore, it is fundamentally different from SDA (NEAR) which is performed using nicking enzymes. It has several additional advantages over such nicking enzyme-dependent methods. For example, there are many more non-nicking restriction enzymes available than nicking enzymes. This means that the restriction enzymes used in the method of the present invention have a large number of potential enzymes. The enzymes are selected from a wide variety of enzymes and are tailored to suit a given application, e.g., reaction temperature, buffer compatibility, stability, and reaction kinetics (sensitivity). This means that it is possible to distinguish between those that have superior properties to ... Because of the significant advantage of this, we have achieved a lower maximum yield than is achievable using nicking enzymes. Restriction enzymes with optimum temperatures and faster kinetics can be selected. is much more suitable for use in low-cost diagnostic devices. The requirement to use restriction enzymes that cut only one strand of their restriction site is in addition to This is an essential feature of the present invention that provides important advantages. For example, the use of specific dNTPs, e.g. The use of thiol dNTPs reduces the melting temperature (Tm) of the DNA into which they are incorporated. In this method, the oligonucleotide primers and probes are generated during amplification. The hybridization sequence of the amplicon is more favorable to the species in the amplified product than to any competing complementary strand containing the modified dNTP. Furthermore, as a result of the insertion of modified dNTP bases, This decreases the Tm of the amplified product, promoting the separation of double-stranded DNA species and thereby increasing the rate of amplification. Alternatively, other modified dNTPs can be used, which enhances the binding of the dNTPs and reduces the temperature optimum, improving sensitivity. This increases the Tm of the incorporated DNA, thereby making it easier to perform the method for a given application. Further opportunities to align are presented.

[0015] The many advantages of the present invention over SDA combine to make the cleavage enzymes more efficient than restriction or nicking enzymes (NEAR). The use of The improved sensitivity and simple visualization of the amplified signal provide the utility of this method.

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

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

[0018] (Detailed Description) The present invention provides a method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample. The target nucleic acid is denatured in the sample, for example, by heat denaturation or through the strand displacement activity of a polymerase. single-stranded DNA from double-stranded DNA after dissociation of the double strands, or RNA, for example, by the activity of reverse transcriptase. The derived single stranded DNA, or, for example, the DNA fragments may be subjected to a nuclease such as a restriction endonuclease or Single-stranded DNA from double-stranded DNA, for example, by the use of enzymes such as Lysosomal enzyme III. Single-stranded DNA, including single-stranded DNA derived from RNA / DNA hybrids via ribonuclease H. The target nucleic acid is obtained by amplifying the DNA in the sample by a DNA polymerase, a helicase, or a recombinase. The single-stranded site in the double-stranded DNA may be, for example, one or more single-stranded DNA from the double-stranded DNA. A transient opening of the DNA base pairs triggers hybridization of the first oligonucleotide primer. By "strand invasion" or DNA fragmentation, which occurs sufficiently to allow extension of the 3' hydroxyl and 3' carboxyl groups. Spontaneous opening of A base pairs, transitional conversion to Hoogsteen pairs, or restriction enzyme or The first step to initiate the method is to productively nick DNA by a thermochemical approach. Sufficiently exposed for hybridization and extension of oligonucleotide primers. The target nucleic acid may be a single strand derived from double-stranded RNA in a sample after dissociation of the two strands, for example by heat denaturation. The RNA may be single-stranded RNA, including double-stranded RNA, or single-stranded RNA derived from double-stranded DNA, for example by transcription.

[0019] The method comprises, in step a) subjecting a sample to: (i) a first oligonucleotide primer and a second oligonucleotide primer; a first oligonucleotide primer, the first primer having a restriction enzyme in the 5' to 3' direction; The recognition sequence and cleavage site and the first hybridization sequence in the target nucleic acid are the second primer comprises one strand of the region capable of hybridizing with the A restriction enzyme recognition sequence and cleavage site in the direction of the first hybridization sequence in the target nucleic acid. The hybridization sequence is then hybridized to the reverse complement of the second hybridization sequence upstream of the hybridization sequence. The first oligonucleotide primer and the second oligonucleotide primer each include one strand of a region capable of being cross-linked with each other. oligonucleotide primers; (ii) strand-displacing DNA polymerase; (iii) dNTPs; (iv) one or more modified dNTPs; (v) a nicking enzyme that recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is blocked by the presence of one or more modifications incorporated into the reverse complementary strand by a DNA polymerase the first restriction enzyme; and (vi) a nicking enzyme that recognizes the recognition sequence of the second primer and and cleaving only the second strand of the primer at the cleavage site if the cleavage site is double stranded. a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by DNA polymerase. The method includes contacting the nucleic acid with a second restriction enzyme,

[0020] If the target nucleic acid to be detected in the sample is double-stranded, either strand may be The target nucleic acid can be considered as a single stranded nucleic acid. It is the product of two oligonucleotide primers. One of them can hybridize to one strand, and the other oligonucleotide This is because the tide primer can hybridize to the other strand. Generally, the oligonucleotide primers used in this method are capable of binding to DNA or RNA targets. DNA primers that form double-stranded DNA or hybrid duplexes containing both strands of RNA and DNA However, primers containing other nucleic acids, such as non-natural bases and / or alternative backbone structures, may be used. Mer may also be used.

[0021] In the presence of the target nucleic acid, the first oligonucleotide primer binds to the first hyaluronan residue in the target nucleic acid. After the hybridization, the first The 3' hydroxyl group of the primer is reacted with a strand displacing DNA polymerase or, optionally, with a target nucleic acid R In the case of NA, the first primer is extended by a reverse transcriptase (e.g., M-MuLV) and the extended first primer is Strand displacement DNA polymerase produces a double-stranded species containing the mer and the target nucleic acid (see FIG. 1). The enzyme or, if present, the reverse transcriptase, uses dNTPs and one or more modified dNTPs in the extension. The restriction enzyme recognition sequence on one strand and the cleavage site at the 5' end of the first primer are typically They do not hybridize normally to the target nucleic acid sequence because their reverse complementary sequences generally Therefore, the first primer generally does not contain a restriction enzyme recognition sequence and The first primer is used to introduce one strand of a cleavage site into the subsequent amplification product species. After extension of the primer, "target removal" occurs. Target removal causes the extended first primer species to , the reverse complement of the second hybridization sequence of the second oligonucleotide primer; When the target nucleic acid is RNA, the target nucleic acid becomes accessible for hybridization. The selective removal of the RNA is accomplished, for example, by RNase H digestion of the RNA, and the RNase H digestion of the reverse transcriptase, if present, is then performed. This can be achieved by the activity of ribozyme H or by the separate addition of this enzyme. Alternatively, if the target nucleic acid is single-stranded DNA containing a single-stranded region in double-stranded DNA, target removal is an additional step. This is achieved by strand displacement using the upstream primer or bump primer. Alternatively, such target removal can be achieved by specifically removing short fragments from a given target nucleic acid molecule. Target removal can occur after spontaneous dissociation when only extension products are produced, or after double-stranded A transient opening of one or more DNA base pairs in the extended first primer species results in a strand-displaced second oligonucleotide. Allows hybridization of nucleotide primers and extension of the 3' hydroxyl This can occur by strand invasion, which occurs sufficiently

[0022] the reverse complement of the second hybridization sequence of the second oligonucleotide primer; After hybridization, the strand-displacing DNA polymerase reacts with the dNTP and one or more modified dNTPs. The 3' hydroxyl of the primer is extended using the first restriction enzyme. A sequence and cleavage site are formed, and one or more modified dNTP bases are incorporated into the reverse complementary strand to form the first It acts to prevent the restriction enzyme from cleaving the strand. The first restriction enzyme does not recognize its recognition sequence. It recognizes the cleavage site and cleaves only the first primer strand, generating a 3' hydroxyl. It is extended by a strand-displacing DNA polymerase using dNTPs and one or more modified dNTPs. The first primer strand is displaced. The second restriction enzyme double-stranded recognition sequence and cleavage site and one or more modified dNTP bases are incorporated into the reverse complementary strand to facilitate pre-amplification by the second restriction enzyme. Therefore, the double-stranded species in which the two primer sequences are juxtaposed is produced, which has partially blocked recognition sites for the first and second restriction enzymes. Then, the first primer strand is cleaved with a first restriction enzyme, and the second primer strand is cleaved with a second restriction enzyme. Restriction enzyme cleavage occurs, producing two double-stranded species, one of which contains the first primer sequence. The first primer strand and the second primer strand are then Sequential cleavage and replacement of mer strands occurs in a cyclic amplification process, where the replaced mer strands are The first primer strand acts as a target for the second primer, and the displaced second primer strand serves as a target for the first primer.

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

[0024] An essential aspect of the present invention is to detect the amplification products of step a) not directly, but by detecting the first and second The oligonucleotide probes hybridize specifically to at least one species in both amplification products. After depolymerization, a detector species is produced. The tide probe is linked to a moiety that allows its detection and detects a first portion of the at least one species. The second oligonucleotide probe hybridizes to the double-stranded detection sequence. is associated with a moiety that allows it to be associated with the solid material, It hybridizes to a second single-stranded detection sequence either upstream or downstream of the first single-stranded detection sequence.

[0025] Those skilled in the art will appreciate by reference to FIG. 1 that the amplification products may be classified into several different species, e.g. For example, the full length or partial sequence or reverse complementary sequence of both the first and second primers. the sequence comprises a primer that binds to a first and a second hybridization sequence in the target nucleic acid. If the primers are separated by one or more bases, they may be separated by target-derived sequences. It will be apparent that the present invention also includes species that contain a single-stranded detection sequence that is The first and second oligonucleotide probes are hybridized to form a detector species. It will be apparent that one may choose to do so.

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

[0027] Two oligonucleotide probes, one for detection and one for binding to the solid material By utilizing this method, the present invention provides rapid and efficient signal detection. This overcomes the need for more complex secondary detection methods, e.g., by nucleic acid lateral flow. This provides efficient visualization of the signal produced in the presence of the target.

[0028] a strand-displacing DNA polymerase for one of the first and second oligonucleotide probes; The present invention relates to a method for the preparation of a nucleic acid sequence which is blocked at the 3' end by the first or second restriction enzyme and cannot be cleaved by the first or second restriction enzyme. Thus, according to a further embodiment, the present invention relates to a method for detecting a defined sequence in a sample. 1. A method for detecting the presence of a single-stranded target nucleic acid in sequence, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; and inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) subjecting the amplification product of step a) to: i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is conjugated to a moiety that enables ligation; wherein one of the first and second oligonucleotide probes is The extension by the DNA polymerase is blocked at the 3' end by the first or second restriction enzyme. and the at least one of the first and second probes in the amplification product is not cleavable by the a detector species is produced by hybridization with said species. and c) detecting the presence of the detector species produced in step b), wherein the presence of a target species indicates the presence of the target nucleic acid in the sample. The method is provided.

[0029] In one embodiment, the single blocked oligonucleotide probe comprises one or more sequences. The above may be achieved by the presence of sequence mismatches and / or one or more modifications, e.g., phosphorothioate linkages. It is incapable of being cleaved by the first or second restriction enzyme. By using a single blocked oligonucleotide probe, it is possible to detect amplification in the presence of a target nucleic acid. Simultaneously with the implementation of step a), i.e., during the production of the product, Thus, according to a further embodiment, the present invention relates to a method for the preparation of a method for the preparation of a sample comprising the steps of: 1. A method for detecting the presence of a single stranded target nucleic acid of a selected sequence comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; and inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) Purifying the amplification product of step a): i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is conjugated to a moiety that enables ligation; wherein one of the first and second oligonucleotide probes is contacted with However, the extension by the DNA polymerase is blocked at the 3' end and the first or second restriction enzyme and the first and second processes are in contact with the sample at the same time as step a) is performed. The probe hybridizes to said at least one species in the amplification product. said contacting resulting in the production of a .actor species; and c) detecting the presence of the detector species produced in step b), wherein the presence of a target species indicates the presence of the target nucleic acid in the sample. The method is provided.

[0030] For example, in the embodiment shown in FIG. 2, the first oligonucleotide probe is blocked, hybridize to at least one species of first single-stranded detection sequence in the amplification product to form a single-stranded region The at least one species is formed to include a 3' hydroxyl group. The DNA polymerase is then extended by a strand-displacing DNA polymerase that extends the predimer group. Thus, in the above embodiment, the blocked oligonucleotide may be stabilized. The oligonucleotide probe contains an additional region, and the blocked oligonucleotide probe is The 3' end of the hybridizing species in the amplification product is extended by the strand displacement DNA polymerase. As shown in Figure 2, a “stable predictor species” is produced. Those skilled in the art will appreciate that this additional predictor in the blocked oligonucleotide probes is The target species stabilization region is a first or second single-stranded detection sequence in at least one species in the amplification product. The blocked oligonucleotide will recognize that the region is upstream of the region that hybridizes with the In embodiments using oligonucleotide probes, blocked oligonucleotide probes and The hybridization sequences of the primers and the appropriate concentrations are determined based on the specific sequences produced in the amplification products. A certain proportion of related species was blocked in each cycle using oligonucleotide probes and hybridization. redistribute the species, and the surviving copies of such species are available to participate in the cyclic amplification process. The oligonucleotide probes can be optimized to remain stable, e.g., by modifying the 3' phosphate. The extension is prevented by the use of a decoration, and in this embodiment, a portion that allows its detection. Alternatively, a single 3' modification can be used to block extension. A variety of other modifications can be used as moieties to allow for the detection of C- 3 spacer, etc., can be used to block the 3' end of an oligonucleotide, Alternatively, mismatched bases can be utilized. The predictor species is ideally The exposed single-stranded region is suitable for efficient detection with a second oligonucleotide probe. The second oligonucleotide is then readily available for hybridization with the second oligonucleotide. The leutide probe is bound to the nitrocellulose surface of the nucleic acid lateral flow strip. This allows the predictor species to be quantified as sequence-specific hyaluronan as it flows through. Hybridization occurs easily and detector species are placed at defined locations on the strip. in the conjugate pad of a nucleic acid lateral flow strip, or Streptavidin-bound carbon, gold, or polystyrene particles that may be present during the amplification reaction Dyes that bind to detection moieties such as , detect the presence of detector species that are produced in the presence of target nucleic acid. Provides quick color-based visualization of

[0031] In another embodiment, the extension by the strand displacement DNA polymerase is blocked at the 3' end. the second oligonucleotide probe being restricted by the first or second restriction enzyme; The second oligonucleotide probe is contacted with the sample at the same time as step a) is performed. The probes are used to prepare the surfaces of electrochemical probes, 96-well plates, and beads before contacting the sample. or binding to a solid material, such as a gel or array surface. At least a certain proportion of the fragments produced during amplification can be combined with a moiety that allows One species hybridizes with the associated reaction primers that further participate in the cyclic amplification process. Rather than generating a nucleotide sequence, the nucleotide sequence is hybridized to a second oligonucleotide probe after its production. After hybridization with the second oligonucleotide probe, the species is A stable predictor species is extended by the polymerase on the nucleotide probe. The first oligonucleotide probe and the detection portion are produced by carrying out step a). The oligonucleotide probes can be simultaneously contacted with the sample and the first oligonucleotide probe can be contacted with the sample at the site of the second oligonucleotide probe. By detecting the accumulation of the detection moiety at the site during the amplification process, This produces a real-time signal that provides quantification of the number of copies of the target nucleic acid present in the sample. Thus, according to an embodiment of the present invention, two or more of steps a), b), and c) are carried out simultaneously. Carry out.

[0032] One of the first and second oligonucleotide probes binds to the DNA polymerase. The extension of the nucleic acid sequence by the first or second restriction enzyme is blocked at the 3' end, and the nucleic acid sequence can be cleaved by the first or second restriction enzyme. In carrying out these embodiments, the present invention provides a method for contacting the sample with the sample simultaneously with carrying out step a). The authors did not observe any significant inhibition of amplification rate and demonstrated that the predictor species was optimal for cyclic amplification. It is shown that the data can be accumulated in real time without disrupting the process. Unequal primer ratios were used to produce one amplicon strand in excess of the other. This contrasts with attempts to operate asymmetric SDA by utilizing blocked oligos. A nucleotide probe is used to remove one amplicon strand from the reaction, thereby Rather than seeking to increase the ratio of the other strand, the present invention seeks to increase the ratio of the other strand during the amplification process. Detector species utilizing blocked probes to promote exposure of single-stranded regions The focus is on production and detection. Therefore, the inventors have Not only did we not observe any inhibitory effect on the process, but in one embodiment, The unexpected increase in generated signal corresponds to at least a 100-fold increase in the amount of detector species. A significant increase in the expression of β-lactamase was observed (see Example 2) (Figure 6).

[0033] Furthermore, one of the first and second oligonucleotide probes is capable of binding to a DNA polymerase. The 3' end of the fragment is blocked from extension by the first or second restriction enzyme, and cannot be cleaved by the first or second restriction enzyme. The sample is contacted with the probe while step a) is being carried out, and the probe is then subjected to a multi-step process without blocking the probe. This study offers fundamental advantages over previous reports of attempts to integrate NEAR with acid lateral flow. For example, WO2014 / 164479 describes a long incubation period of 30 minutes at 48° C. Incubation is required to visualize the amplification products using nucleic acid lateral flow. This has been the case in point-of-care diagnostic devices, especially low-cost or disposable devices. In stark contrast, the method of the present invention has a lower intensity. Equivalent amplification is easily achieved within 5 minutes at incubation temperatures of, for example, 40-45°C. In a direct comparative study (see Example 10), the method of the present invention uses a nicking enzyme Use of restriction enzymes other than dNTPs, use of modified dNTP bases, and blocked oligonucleotides Compared to the prior art method (WO2014 / 164479) which resulted from the combined use of probes, It shows extremely good speed without any problems.

[0034] The other of the first and second oligonucleotide probes is The extension by the enzyme is blocked at the 3' end by the first or second restriction enzyme described above. Please be aware that you cannot disconnect it.

[0035] An essential aspect of the method is not the nicking enzyme, but the recognition sequence and cleavage site are The reverse complementary strand is double stranded and cleavage of the reverse complementary strand is accompanied by incorporation of one or more modified dNTPs, e.g. The above is synthesized by strand-displacing DNA polymerase using dNTPs that confer nuclease resistance to the subsequent Recognition of its recognition sequence when blocked by the presence of one or more modifications incorporated into the reverse complementary strand and the use of one or more restriction enzymes that can cleave only one strand of the cleavage site. .

[0036] A "restriction enzyme" [or "restriction endonuclease"] binds to a specific recognition sequence and then A nucleic acid molecule is synthesized by cleaving one or more phosphodiester bonds in one or both strands of the double-stranded nucleic acid molecule at a predetermined cleavage site. A wide variety of restriction enzymes are available, with over 3,000 reported. There are over 600 commercially available, each with a wide variety of different physicochemical properties and recognition sequence characteristics. The opposite sex is covered.

[0037] A "nicking enzyme" (or "nicking endonuclease") is a specific subunit of a restriction enzyme. class of nucleic acid fragments that, after binding to a specific recognition sequence, cleave one side of a double-stranded nucleic acid molecule at a specific cleavage site. Only one strand can be cleaved, leaving the other strand intact. Only binding enzymes are available, both natural and engineered. The king enzymes are the bottom strand cutters Nb.BbvCI, Nb.BsmI, and Nb.B srDI, Nb.BssSI, and Nb.BtsI and the top strand cutters Nt.AlwI, These are Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.

[0038] The non-nicking restriction enzymes utilized exclusively in the methods of the present invention are double-stranded Although it can cleave both strands of nucleic acid, it does not specifically bind to its recognition sequence. Under certain circumstances, it breaks or nicks only one strand of the double-stranded DNA break site. This can be achieved in several ways. Of particular relevance to the present method are: This means that one of the strands in the double-stranded nucleic acid at the cleavage site is repaired while one of the strands in the double-stranded nucleic acid target site is repaired. The phosphodiester bond at the cleavage site on one strand is modified so that the nuclease Resistant modifications, such as phosphorothioates (PTO), boranophosphates, methyl phosphates They cannot be cleaved because they are protected by a peptide-nucleotide bond or a peptide-nucleotide bond. This can be achieved when certain modified internucleotide linkages, such as PT O-linkages can be provided in oligonucleotide probes and primers by chemical synthesis. Alternatively, for example, one or more α-thiol modified deoxynucleotides can be used to prepare a polymerase Thus, in one embodiment, one or more The modified dNTP is an α-thiol modified dNTP. Typically, it is incorporated to confer nuclease resistance. The S isomer is used because it is more effective.

[0039] The large number of non-nicking restriction enzymes available has different properties. A wide variety of enzymes can be selected based on desired performance characteristics, e.g., for use in a method for a given application. of, temperature profile, speed, buffer compatibility, polymerase cross-compatibility, recognition sequence, thermal can be used to screen for stability, manufacturability, etc. In contrast, very few Due to the fact that only a few nicking enzymes are available, conventional techniques using nicking enzymes The potential of surgical methods is limited, e.g. by lower reaction rates (sensitivity, time to results). and higher reaction temperatures may result. The non-King restriction enzyme may be a naturally occurring or engineered enzyme.

[0040] In selecting a restriction enzyme that is not a nicking enzyme for use in this method, one skilled in the art If so, the modification is incorporated at the appropriate position to prevent cleavage of the relevant strand and inhibit cleavage of the other strand. To ensure that cleavage is not inhibited, it is necessary to identify an enzyme with an appropriate cleavage site. For example, modified dNTPs, such as α-thiol dNTPs, may be used. In certain embodiments, sufficient flexibility in primer placement is provided to allow for target nucleic acid sequences to be identified. contains a modified nucleotide base at the appropriate position to prevent cleavage of the associated strand after its incorporation In order to allow the recognition sequence to be determined, it is necessary to use an asymmetric restriction enzyme having a non-palindromic recognition sequence. It may be preferable to choose a restriction enzyme that has a cutting site outside the sequence. If all-dATP is used, ensure that the primer is properly cleaved during the procedure. To ensure that the sequence is consistent with the reverse complement of the restriction enzyme cleavage site of the relevant oligonucleotide primer, The sequence includes an adenosine base downstream of the cleavage site in the reverse complementary strand, but does not include the primer sequence It does not include the adenosine base downstream of the cleavage site in the Asymmetric restriction enzymes that have non-palindromic recognition sequences that cut outside of the target region are suitable for use in the present invention. It is suitable for use in recognizing partial or degenerate palindromic sequences that cleave within their recognition sites. Restriction enzymes that are suitable for use in the present invention may also be used. Nuclease-resistant nucleotide linkage modifications, e.g. PTO allows the detection of any of a wide variety of commercially available double-strand breakers from a variety of different classes. One such class of double-strand breakers is the Partial or degenerate palindromes and asymmetric sequences to enable their use in the disclosed methods. There are two types of restriction enzymes, type IIS and type IIG, which have both identical restriction enzyme sequences.

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

[0042] In one embodiment, the first and second restriction enzymes are the same restriction enzyme. By using only 1,000,000 dimethylformamide, the process is simplified in many ways. A single suitable enzyme is identified and optimized for the implementation of the method, and the production and stability of the enzyme are evaluated. In addition, the use of a single restriction enzyme allows for the synthesis of oligonucleotide primers. This simplifies the design of the immersion and supports symmetry of the amplification process.

[0043] In the method, the restriction enzyme cuts only one strand of the nucleic acid duplex, and therefore after cutting, These have exposed 3' ends that can act as efficient polymerase priming sites. The polymerase extends the primer and forms a base pairing interaction. A strand or polymer of nucleic acid is synthesized by generating a reverse complementary copy of a DNA or RNA template strand using Polymerases with strand displacement ability can displace the strand appropriately to complete the amplification process. The term "strand displacement" refers to the effect of a strand displacement reaction on the synthesis of a nucleic acid molecule. This refers to the ability of a polymerase to displace downstream DNA it encounters. A variety of polymerases with this ability have been characterized and are commercially available. i29 polymerase has a strong strand displacement ability. Polymerases from Bacillus species, e.g. For example, the Bst DNA polymerase large fragment typically exhibits high strand displacement activity and is useful in carrying out the method. The E. coli Klenow fragment (exo-) is widely used. Strand displacement polymerases, such as KlenTaq, are another type of strand-displacing polymerase that Cloning of only the relevant active polymerase domain of the current enzyme and any exonuclease The target nucleic acid can be easily manipulated by knocking out the enzyme activity. For the implementation of the method in which the acid is RNA, RNA-dependent DNA synthesis (reverse transcriptase) activity is also required. This activity can be achieved by a strand displacing polymerase and / or by a separate additional reverse transcriptase in step a). This can be achieved by using a gene such as M-MuLV or AMV.

[0044] Polymerases are typically used in conjunction with enzymes, reagent concentrations, and desired For example, Bacillus polymerase is used in an appropriate amount optimized according to the reaction temperature. 0.1 to 100 units of enzyme can be used; 1 unit is required to oxidize 25 nmol of dNTPs for 30 minutes at 65°C. It is defined as the amount of enzyme that is incorporated into the insoluble material. However, the amount can vary depending on several factors, e.g. For example, polymerase activity, its concentration and form, expected concentration of target nucleic acid, reaction volume, oligonucleotide The number and concentration of the primers and the reaction temperature are the factors that determine the reaction time. should not be considered as determining

[0045] Those skilled in the art will appreciate that a polymerase requires dNTP monomers in order to have polymerase activity. The polymerase also contains components such as buffer salts, divalent ions, and stabilizers. In addition, strand-displacing polymerases are known to require appropriate buffers. To prevent cleavage of the reverse complement of the primer after incorporation, one or more modified dNTPs are used in this method. Typically, when a single modified dNTP is used, the dNTP used in this method is For example, in an embodiment where the modified dNTP is an α-thiol dATP, In this case, dNTPs include only dTTP, dCTP, and dGTP, and do not include dATP. By removing the group, only the modified base becomes available for incorporation by the polymerase, All desired bottom strand cleavage sites in the reverse complementary sequence of the primer are blocked. However, complete or partial removal of the corresponding natural dNTP bases is not essential. dNTPs are typically used in this method at concentrations not typically used in other polymerase methods. Concentrations as high as 10 micromolar to 1 millimolar may be used, but should not be exceeded. Maximize activity and minimize synthesis from 1 to avoid generating background signal For the purposes of this method, the dNTP concentration is set for any given enzyme and reagent. It can be optimized to detect whether a particular polymerase reacts with one or more modified dNTP bases at a slower rate. Considering that one or more modified bases may exhibit incorporation of dNTPs with higher correlation than unmodified dNTPs, Concentrations, for example 5-fold higher, can be used in this method, but this is not a limiting example. should be regarded as something.

[0046] The use of one or more modified dNTPs causes restriction enzymes to cleave only one strand at their restriction site. In addition, it is an essential feature of the present invention that provides important advantages. The use of TPs, such as α-thiol dNTPs, reduces the melting temperature (Tm) of the DNA into which they are incorporated. This is because the oligonucleotide primers and probes used in this method However, it is believed that the dNTPs produced during amplification are more likely to form hybrids with the species in the amplification product than with any competing modified dNTP complements produced during amplification. This important feature means that the For example, one of the displaced strands may hybridize with its reverse complement to produce a "non-productive" end species. When the nucleic acid is produced, the presence of one or more modified bases results in a decrease in the Tm of hybridization. This allows for "productive" hybridization of the displaced strand with a further primer. Since phosphorothioate nucleotides dissociate more easily than cations, the rate of amplification is enhanced. The interstrand bond increases Tm (the temperature at which exactly half of the double strands hybridize together). It has been reported that this can lower the temperature by 1 to 3 degrees Celsius and significantly change the physicochemical properties. The inventors have demonstrated that the presence of phosphorothioate nucleotide bonds in DNA sequences increases the strand displacement rate. Furthermore, the oligonucleotide probe used in this method Regardless of whether the sample is contacted with the sample simultaneously with the implementation of step a) or after step a), has a higher affinity for the species in the amplification product than any competing modified species, and therefore preferentially or even displace the hybridized strand to form a detector species. It contains modified internucleotide bonds that can enhance the production of amplified species with reduced Tm. The increased number of substitutions and substitutions radically increases the speed of the method, and is necessary for rapid amplification to occur. It helps to lower the temperature.

[0047] In addition to the rate enhancement through the use of one or more modified nucleotides, oligonucleotide primers can be used to The hybridization specificity of the mers and probes of this method is also enhanced. Assuming that all bases of one particular nucleotide are replaced in the amplification product, The hybridization sites of primers and probes typically contain modified bases and The decrease in Tm due to phosphorothioate internucleotide bonds can be attributed to, for example, non-specific hybridization. This means that sequence mismatches due to amplification are less likely to be tolerated.

[0048] Thus, the essential feature of the method of the invention of one or more modified dNTPs is the sensitivity and specificity of the amplification. Both methods are known, e.g., software-based methods that enhance the A modification of NEAR (WO2014 / 164479) using primers optimized by software or Warmth The same applies to NEAR (WO2009 / 012246), including start-up or controlled temperature reduction (WO2018 / 002649) This brings fundamental advantages that are starkly contrasting.

[0049] Several different modified dNTPs, which are nuclease-resistant after their incorporation by polymerases In one embodiment, modified dNTPs are present that confer resistance to cleavage by a restriction enzyme and, in one embodiment, Other features that can be used in the method to enhance the performance of the method for a given application may be achieved. In addition to the α-thiol dNTPs that provide nuclease resistance and a lower Tm, Modified dNTPs that have been reported to have the potential for enzyme incorporation and confer nuclease resistance These include equivalent bases such as borano derivatives, 2'-O-methyl (2'OMe) modified bases and 2'-fluoro bases. There are nucleotide derivatives that are incorporated by the polymerase and provide specific properties of the method. Other modified dNTPs or equivalent compounds that may be used in embodiments of the method for enhancing , which reduces binding affinity, e.g., inosine-5'-triphosphate or 2'-deoxyglucose 5'-triphosphate, those that increase binding specificity, e.g. 5-methyl-2'-deoxycytidine 5'-triphosphate, or 5-[(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine -5'-triphosphate, and those that enhance the synthesis of GC-rich regions, such as 7-deaza-dGTP. Certain modifications can increase the Tm and, in embodiments of this method, This further offers the potential to control elimination events.

[0050] Steps a), b) and c) can be carried out over a wide range of temperatures. The optimum temperature for each step is The optimum temperatures for the relevant polymerases and restriction enzymes, as well as the oligonucleotide primers The melting temperature of the hybridizing region of the nucleotide sequence is determined by the melting temperature of the nucleotide sequence. The present invention does not use temperature cycling in step a). Furthermore, the amplification step a) does not involve a controlled increase in temperature. reduction, hot or warm start, preheat, or controlled temperature reduction The method can be carried out in a wide range of temperatures, for example from 15°C to 60°C, for example from 20°C to 60°C or The steps can be carried out at temperatures ranging from 15 to 45° C. According to an embodiment, step a) is carried out at temperatures below 50° C. or at a temperature of about 50° C. Various non-nicking enzymes are available for use in this method. Considering the wide variety of restriction enzymes available, an alternative method using nicking enzymes is It is possible to choose restriction enzymes that have rapid kinetics at relatively low temperatures in comparison. Additionally, the use of one or more modified nucleotides reduces the temperature required for amplification. In addition to having the potential to lower the optimal temperature profile compared to known methods, The method of the present invention can be carried out over an exceptionally wide temperature range. This makes it very attractive for this method to be used in low-cost diagnostic devices where the heating control is disposable. The cost of goods for such devices to the point where devices that eliminate the need for equipment or devices are not commercially practical. For example, the rapid synthesis of target nucleic acids at ambient temperature or approximately 37° C. Several assays have been developed using methods that can perform detection. Thus, in a further embodiment, step a) is carried out at a temperature below or at about 45° C. To simplify the user process and reduce the overall time to results, It may be preferable to start the process at a lower temperature. In an embodiment, the temperature of step a) is increased during amplification. For example, the temperature of this method is increased to ambient temperature. The temperature is increased over a period of time, for example, 2 minutes, starting at, for example, 20° C., and the final temperature, For example, it reaches approximately 45° C. or 50° C. In one embodiment, the starting ambient temperature is, for example, 15 to 30° C. Increasing the temperature during the implementation of step a), such as increasing the temperature from a range of 40° C. to a range of 40-50° C. do.

[0051] The low temperature potential and versatility of the method of the present invention make it possible to achieve a high yield in a wide range of applications. In particular, immunoassays or assays for detecting other biomarkers such as proteins or small molecules. This means that it is compatible with the requirements of a variety of other assays, including enzyme assays. Thus, the method can be used to identify, for example, both nucleic acids and proteins or small molecules of interest in a sample. It can be used for detection at the time of DNA fragmentation. enzyme, oligonucleotide primer, oligonucleotide probe, dNTP and one or more The components required to carry out this method, including the modified dNTPs, are lyophilized for stable storage. or freeze-dried, and the reaction is subsequently triggered by rehydration, e.g., upon addition of sample. Such stable freeze-drying or lyophilization typically involves one or more It may be necessary to add an excipient, such as trehalose, before drying the ingredients. A wide variety of such excipients and stabilizers for freeze-drying or lyophilization are known. and is available for testing to identify suitable compositions of components required to carry out this method. do.

[0052] Those skilled in the art will appreciate that the method of the present invention is a polymerase-based amplification method, and may be performed using PCR or other polymerases. Addition of one or more additives that have been shown to enhance polymerase-based amplification methods It will be apparent that the additives can be further enhanced. Such additives include, but are not limited to, However, tetrahydrothiophene 1-oxide, L-lysine free base, L-arginine, glycine histidine, 5-aminovaleric acid, 1,5-diamino-2-methylpentane, N,N′-diisopropyl Diethylendiamine, tetramethylenediamine (TEMED), tetramethylammonium chloride , tetramethylammonium oxylate, methylsulphate Phenacetamide, Hexadecyltrimethylammonium bromide, Betaine aldehyde, Tetradecyl Tetraethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride ammonium chloride, tetrabutyl ammonium chloride, tetrapropyl ammonium chloride, dimethylformamide (DMF), N-methylformamide, N-methylacetamide , N,N-dimethylacetamide, L-threonine, N,N-dimethylethylenediamine, 2-pyrrolidin Don, HEP (N-hydroxyethylpyrrolidone), NMP (N-methylpyrrolidone), and 1-methyl , 1-cyclohexyl-2-pyrrolidone (pyrrolidinone), δ-valerolactam, N-methylsuccinimide imide, 1-formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose These include glycerol, Tween-20, DMSO, betaine, and BSA.

[0053] Our studies have demonstrated that the method can detect very low copy numbers, even single copy numbers. The results showed that the oligonucleotides are effective over a wide range of target nucleic acid levels. The primers are typically provided in large excess to the target nucleic acid. Typically, the concentration of each primer is is in the range of 10-200 nM, but should not be considered as limiting. Primers Higher concentrations can enhance the efficiency of hybridization and thus increase the reaction rate. However, non-specific background effects such as primer dimers were observed at high concentrations. Therefore, the concentrations of the first and second oligonucleotide primers may be varied using this method. This forms part of the optimization process for any given assay. In another embodiment, the first and second oligonucleotide primers are provided at the same concentration. In the method, one of the first and second oligonucleotide primers is Due to the natural symmetry of the cyclic amplification process, one primer is provided in excess of the other. Although it is often provided in excess of the other, in certain circumstances it may be possible to reduce the non-specific background of the method. reducing the interdigital signal and / or improving the hybridization of the first and second oligonucleotide probes. Implementations that can be used to enhance the ability to produce detector species by resizing In embodiments, the rate of the reaction may be slowed down. Both primers are present at non-limiting levels before the target species is produced. It is desirable.

[0054] To improve the performance of this method, several considerations must be considered in the design of the oligonucleotide primers. The first and second oligonucleotide primers each have a 5' to 3' sequence It must contain a restriction enzyme recognition sequence and cleavage site as well as one strand of the hybridizing region. In the case of the first primer, the hybridization region is the first hybridization region in the target nucleic acid. In the case of the second primer, the first hybridization region in the target nucleic acid is The hybridization sequence is a reverse complement of the second hybridization sequence upstream of the hybridization sequence. Thus, a pair of primers is designed to amplify a region of a target nucleic acid. The restriction enzyme recognition sequence of the primer is typically not present in the target nucleic acid sequence. After the hybridization event, overhangs are formed and introduced into the amplicon. (See FIG. 1.) When asymmetric restriction enzymes are used, the cleavage site is typically It is downstream of the sequence and therefore may optionally be present in the hybridizing sequence of the primer.

[0055] The oligonucleotide primer, after its cleavage in this method, has a sequence 5' to the cleavage site. , which continues to hybridize with its reverse complementary strand under desired reaction conditions and forms a strand-displacing DNA polymerase A melting temperature sufficient for the enzyme to displace the strand downstream of the cleavage site after extension of the 3' hydroxyl group. The upstream primer is designed to have a temperature (Tm) which provides additional "stabilization" A "" region may be included at the 5' end of the oligonucleotide primer. Its optimal length is The length is determined by the location of the cleavage site relative to the recognition sequence of the relevant restriction enzyme and the length of the restriction enzyme that is useful for the amplification in step a). It is determined by other factors such as the temperature used. and / or the second oligonucleotide primer is The stabilizing sequence may include a stabilizing sequence upstream of the nucleotide sequence, for example at the 5' end, and a stabilizing sequence, for example 5 or 6 bases in length.

[0056] Primer design allows optimal sequence-specific hybridization and strand displacement. To ensure specific and sensitive amplification in this method, The sequence and length must be defined. The target nucleic acid to be detected, e.g. a viral or bacterial pathogen, The positioning of the primers in the genome of the organism varies the sequence of the hybridizing region of the primers. The amplification sensitivity and specificity are therefore optimized and the compatibility with oligonucleotide probes is improved. Therefore, different primer pairs can be screened. It is possible to identify optimal sequences and positions to enhance the performance of this method. Typically, the length of the hybridizing region of the primer is determined so that its theoretical Tm is equal to or greater than the desired Tm. It allows efficient hybridization at the reaction temperature, but at the same time allows easy replacement after cleavage. During primer design, the hybridizing sequence and the displaced strand are designed. The theoretical Tm of the sequence is considered under the expected reaction temperature and the conditions of the selected restriction enzyme. This is balanced against the theoretical improvement toward sequence-driven binding specificity that may occur with increasing sequence length. Through various studies by the present inventors, it has become possible to design primers that can be effectively used in this method. The scheme has been shown to have considerable versatility. In one embodiment, the first and / or second The hybridization region of the oligonucleotide primer is 6 to 30 bases long, for example 9 to 16 bases long. In further embodiments, modifications, such as unnatural bases and alternative nucleotides, The hybridization region of the primer can be modified to accommodate specific applications by utilizing the inter-linked junction or abasic site. Therefore, their properties and the operation of this method can be refined. For example, to increase the Tm Modifications such as PNA, LNA, G-clamp make the amplicons shorter and therefore enhance the rate of amplification This allows for shorter and more specific primer hybridization regions that can be possible.

[0057] Our various studies have demonstrated that the speed and sensitivity of this method are excellent for detecting short amplicons. and thus in one embodiment includes the hybridizing sequence. By shortening the overall length of both primers and by determining whether the first and second hybridization sites of the target nucleic acid are identical, There is only one short gap between the sequences, e.g., 10 or 15 nucleotide bases or less. It has become apparent that it may be preferable to position the primer such that In the embodiment, the first and second hybridization sequences of the target nucleic acid are 0 to 15 or 0 to 6 bases long. In certain embodiments, they are separated by 3 to 15 or 3 to 6 bases, e.g., 5, 7, or 11 In further embodiments, the hybridization occurs by, for example, one or two bases. The amplification sequences are overlapping.

[0058] There are several considerations in designing the sequence of an oligonucleotide probe for use in this method. First, a first oligonucleotide that hybridizes with a first single-stranded detection sequence is A second oligonucleotide that hybridizes to a region of the oligonucleotide probe and a second single-stranded detection sequence. The regions of the nucleic acid probes are typically spaced such that they do not overlap or have minimal overlap. and both oligonucleotide probes have a double overlap and both oligonucleotide probes are capable of detecting at least one species in the amplification product. and to one or more species in the amplification product. Both oligonucleotide probes bind to the same strand, ensuring efficient targeting. To achieve this, they typically focus primarily on the location of the cleavage site on one strand of the amplified product species and vice versa. Designed to hybridize to a sequence between the positions on the complementary strand opposite the cleavage site For any given primer pair, both strands are probed with oligonucleotide probes. Oligonucleotide probes are typically used for this purpose. Considering that the hybridization sequence is not extended by polymerase in the method, The products are designed based on the relative sequences of the species in the product, which determines their Tm, %GC, and the resulting In one embodiment, the first and / or second oligonucleotides are The hybridization sequence of the tide probe is 9 to 20 nucleotide bases in length. and the second hybridization sequence are separated by 0 bases. The sequence of the hybridizing region of one of the oligonucleotide probes is It may correspond to one of the oligonucleotide primers and the other The hybridizing region of the first strand corresponds to the reverse complement of the other oligonucleotide primer. However, the characteristics of the oligonucleotide probe for the desired embodiment of this method are Optimize to eliminate any inhibitory effects when all or part of step b) is carried out simultaneously with step a). To avoid this, the length of the hybridizing sequence can be shortened. The nucleotide probe includes a recognition sequence and a cleavage site of a first or second restriction enzyme, When the oligonucleotide probe is contacted with the sample simultaneously with carrying out step a), The cleavage site may be, for example, a modified internucleotide bond, e.g., a phosphoro Including thioate bonds or introducing mismatches to remove the recognition sequence In addition to the hybridization region, oligonucleotide probes are typically used. The sequences of the lobes and any modified nucleotide bases, nucleotide linkages, or There is considerable versatility in other modifications that can be chemically inserted into oligonucleotides to modify their Modified bases that have altered properties and can be utilized in embodiments of this method, such as 2-amino-dA. , 5-methyl-dC, Super T®, 2-fluoro base, and G clamp provide an increase in Tm. While other modified nucleotide bases, such as Iso-dC and Iso-G, do not increase the Tm, Other modifications, such as inosine or abasic sites, can enhance binding specificity. Modifications known to confer nuclease resistance include inverted dT and The modifications can increase or decrease the Tm, and this method In embodiments, this provides the potential for controlling hybridization events. The use of modified bases in the hybridizing region of a nucleic acid probe can, for example, By increasing the binding affinity of the oligonucleotide domain without increasing its length, In one embodiment, one or both of the The modified bases in the oligonucleotide probes have complementarity with the associated single-stranded detection sequence. hybridize more efficiently, and therefore outcompete, any species in the amplification product that This allows for the

[0059] One of the first and second oligonucleotide probes is blocked from extension at the 3' end and cleaved. In an embodiment in which the sample is contacted simultaneously with the performance of step a), the Generally, one oligonucleotide probe contains an additional 5' region and is pre-assembled as described. This provides an opportunity to stabilize the detector species (see FIG. 2). In the method, the one oligonucleotide probe is a nucleic acid sequence of an oligonucleotide primer. It contains the exact sequence of one of these but blocks its extension by strand-displacing DNA polymerase at the 3' end. A single phosphorothioate internucleotide linkage blocks modification and restriction enzyme cleavage sites. Such embodiments simplify assay design and reduce non-specific background enhancement. Be sure not to introduce additional sequence motifs that could result in width.

[0060] The first and second oligonucleotide probes which produce the detector species are preferably The copy number of the detector species produced is utilized for the detector species that is easy to detect. In addition, the first and / or second oligonucleotides are provided at levels well above the detection limits of the means for detecting the presence of the oligonucleotide. The efficiency of hybridization with a oligonucleotide probe is affected by its concentration. Typically, the concentration of oligonucleotide probe contacted with the sample simultaneously with carrying out step a) is The concentration of the oligonucleotide primer may be about the same as that of the oligonucleotide primer, for example, 10 to 200 nM. This should not be considered limiting. In one embodiment, one or both The concentration of the oligonucleotide probe is determined by the concentration of one or both of the oligonucleotide primers. In another embodiment, one or both of the oligonucleotides are provided in excess of the The concentration of the oligonucleotide probe is lower than that of one or both of the oligonucleotide primers. After carrying out the amplification step a), one or both of the oligonucleotide probes are When contacting with a soluble form of β-lactam, a higher concentration may be used to achieve the most efficient hybridization, if desired. This makes it possible to achieve amplification and thus to prevent amplification that may occur as a result of step a). There is no need to consider any inhibition.

[0061] The hybridization sequences are oligonucleotide primers for carrying out this method. This is an important feature of both oligonucleotide and nucleotide probes. refers to an oligonucleotide primer or probe that binds to a species in a target nucleic acid or an amplification product. This refers to the ability of a nucleic acid molecule to hybridize to a specific base pair in a nucleic acid sequence. A typical base pair is adenine-thymine (AT), or RNase A. A or RNA / DNA hybrid duplexes, adenine-uracil and cytosine-guanine Although the majority of nucleotide analogs are nucleotides, various natural and unnatural analogs of nucleobases have also been shown to have specific binding preferences. Furthermore, in the present invention, the phase of the oligonucleotide probe or primer A complementary region does not necessarily correspond to its hybridization sequence in a species of the target nucleic acid or amplification product. It is not necessary to have completely natural nucleobases in a sequence that has complete and exact complementarity to the bases of the nucleic acid. However, for the practice of this method, the oligonucleotide probe / primer is Required for the correct operation of this method, which involves cleavage by and extension by strand-displacing DNA polymerase and a sequence-specific hybridization sequence with its target sequence sufficient to form a double-stranded sequence. It is sufficient that hybridization is possible. Synthesis can occur even if the nucleic acid is not exactly complementary and has unnatural bases or abasic sites. In one embodiment, the oligonucleotide primer or oligonucleotide used in this method The hybridizing region of the oligonucleotide probe is a region that hybridizes with the sequence of the relevant region of the target nucleic acid. It may consist of a perfect complement, a species in the amplified product, or, where appropriate, a reverse complement thereof. In some embodiments, there are one or more non-complementary base pairs. It is advantageous to use a mixture of oligonucleotide primers and / or probes in the Thus, for example, a single nucleotide polymorphism (SNP) site may be present where the target nucleic acid has two polymorphic positions. If the oligonucleotide primer contains a different oligonucleotide primer at that position, A 1:1 mixture of lobes (each component having complementarity to each base of the SNP) can be used. When an oligonucleotide is produced, one or more bases can be randomized during the synthesis process. is customarily practiced.

[0062] Those skilled in the art will appreciate that the amplification process involving a polymerase is a step in the synthesis of a first and / or second primer. It is understood that non-specific background amplification, such as that caused by inter-binding, can be an issue. The methods of the invention typically involve shortening the length of the amplicon as much as possible, e.g. For example, the hybridization sequence of the primer, the first and second hybridization sequences in the target nucleic acid, Minimize gaps between the transcription sequences and the length of any stabilizing regions to the extent possible. shows faster amplification when designed with With shorter amplicons, the required length of the amplicon needed to produce the amplified species is still retained. The fact that all the necessary sequences are provided by the oligonucleotide primers makes it possible to The specific background may worsen. A first oligonucleotide primer and a second oligonucleotide primer "linked" via ligation. If an amplicon is produced in a non-target specific manner that contains both the target primers, a false positive is considered. In this method, two oligonucleotide probes are used. The use of such a method allows for the inclusion of additional features of various embodiments, and allows for the inclusion of any non-target The possibility of specific background signals is minimized. Such an embodiment, which is realized by using a probe, is significantly superior to known methods in this respect. There are great advantages to this.

[0063] One approach involves separating the first and second hybridization sequences in the target nucleic acid. To perform target-based sequence specificity checks using oligonucleotide probes in this method, Thus, in one embodiment, a first and a second hybridization of a target nucleic acid is provided. The amplification sequences are separated by 3 to 15 or 3 to 6 bases, for example, 5, 7, or 11 bases. This gap between primers represents an optimal size gap to allow for rate enhancement of short amplicons. It provides an additional check on the specificity of the species in the amplified product while maintaining robustness. In the above, in step b), the first or second nucleotide of the at least one species in the amplification product is The single-stranded detection sequence has a sequence of at least 3 bases corresponding to the 3 to 15 bases or the 3 to 6 bases. For example, the present inventors have demonstrated that specific target-dependent amplification products and We demonstrated the potential to discriminate between target-specific and non-target background amplification products.

[0064] In an alternative approach, the concentration of the first and / or second oligonucleotide primers is reduced. This reduces the probability of background amplification from 1 and primer binding. To ensure that the amplification rate is maintained, the extension with strand-displacing DNA polymerase is repeated for three Additional oligonucleotide primers blocked at the ends can be used. In an embodiment, the first and second oligonucleotide primers are not blocked. A single hybridization and extension event is performed to produce an amplicon derived from the target nucleic acid. is preferably available in sufficient concentration to inhibit subsequent amplification, The cleavage of the blocked primer is then followed by the 3' blocking modification. and before its extension and strand displacement in order to allow the amplification process to continue without loss. (See FIG. 4). Thus, in one embodiment, in step a) the sample is Further, (A) in the 5' to 3' direction, the recognition sequence and cleavage site of the first restriction enzyme and the target A region in the target nucleic acid capable of hybridizing to the first hybridization sequence a third oligonucleotide, which contains one strand of the and / or (B) a recognition sequence for the second restriction enzyme in the 5' to 3' direction; The cleavage site and the reverse complement of the second hybridization sequence in the target nucleic acid are hybridized. and the DNA polymerase is capable of extending the 3' end of the region that can be modified. The nucleic acid sequence is contacted with a fourth oligonucleotide primer, the fourth oligonucleotide primer being blocked at the end. wherein, if present, the third oligonucleotide primer is provided in excess of the nucleotide primer and, if present, the fourth oligonucleotide The oligonucleotide primer is provided in excess of the second oligonucleotide primer. and the second oligonucleotide primer were significantly reduced in concentration, and this was combined with the third and fourth oligonucleotide primers. Non-target dependent batches are generated by compensating for the presence of oligonucleotide primers. This provides the greatest potential benefit in terms of elimination of background amplification. - Polymerases that can be easily achieved during synthesis, for example, through the use of 3' phosphate or C-3 modifications Utilized for the first and second primers, except for the presence of a 3' modification to prevent extension. The same design parameters apply to the third and fourth primers.

[0065] As described above, the practice of the methods of the invention provides enhanced specificity and elimination of background amplification. The embodiment improves the stringency of sequence confirmation, thereby allowing lower temperature reactions to be performed without loss of specificity. and / or increase multiplexing, allowing multiple reactions to be performed in multiple The advantage of this strict specificity is that it can be performed with simultaneous detection of targets. The method also loses specificity over a wide temperature range and at suboptimal conditions (e.g., reagent concentrations). For example, the inventors have reduced the concentrations of all ingredients by 20%. The inventors carried out the method by increasing and decreasing the number of times, and after carrying out the amplification in step a), The method was carried out at ambient temperature and in each case no loss of specificity was observed. Such an embodiment is therefore an important advantage of the method of the present invention over known methods. This means that they are ideally suited for use in low-cost and / or disposable diagnostic devices. do.

[0066] The detection of the detector species in step c) involves isolating the detector species from other reagents and components present in the sample. This can be accomplished by any technique that differentially detects the presence of a vector species. The presence or level of the detector species may be determined by one or more reaction components, such as a first or second oligonucleotide. It can be deduced from the removal of nucleotide probes. Available for use in detecting detector species. The first and second oligonucleotide probes can be prepared from a wide variety of physicochemical techniques available. It exists only after hybridization of the nucleotide probe with the related species in the amplification product. Existing technologies capable of generating highly sensitive signals were prioritized for use in this method. Those skilled in the art can easily determine the sequence of the first oligonucleotide probe that can be attached to the first oligonucleotide probe. There are a variety of colorimetric or fluorometric dyes available that can be measured visually or using instruments (e.g. It will be apparent that such techniques will form the basis of detection (e.g., absorbance or fluorescence spectroscopy).

[0067] Thus, in one embodiment, a method for detecting the first oligonucleotide probe is provided. The portion that is to be coated is a colorimetric or fluorometric dye, or a combination of a colorimetric or fluorometric dye. A moiety that allows for binding, such as biotin.

[0068] Method embodiments utilizing colorimetric dyes do not require equipment for fluorescence excitation and detection. Colorimetric detection has the advantage that the presence of the target nucleic acid can potentially be determined visually. Prior to its use in the method, the first oligonucleotide probe is provided with a colorimetric dye or a colorimetric dye. A moiety capable of binding to a colorimetric dye is directly bound to the probe, or a dye or a probe fragment is bound to the probe after cleavage. This is achieved by specifically attaching or binding the moiety. For example, the first oligonucleotide probe may be provided with a second oligonucleotide probe that can subsequently be used to detect it. The resulting dye is then conjugated to streptavidin to allow its binding to a colorimetric dye for detection. An example of such a colorimetric dye that can be used for detection is a gold nanoparticle. A variety of other essentially colorimetric moieties can be utilized in a similar manner. Numerous types of nanoparticles are known, such as carbon nanoparticles, silver nanoparticles, and nanoiron oxide nanoparticles. Examples include nanoparticles, polystyrene beads, and quantum dots. Dyes with high extinction coefficients are also This offers the potential for highly sensitive real-time quantification in a method.

[0069] Several considerations are taken into account when selecting an appropriate dye for a given application, for example: In embodiments intended for performing visible colorimetric detection in solution, one In general, it is advantageous to select larger sized particles and / or particles with a higher extinction coefficient. While this is advantageous, in embodiments incorporating lateral flow membranes intended for visual detection, may benefit from the ability of smaller sized particles to diffuse more rapidly along Gold nanoparticles are available in a variety of sizes and shapes, but are typically coated on polystyrene or latex. Some other colorimetric assays of interest include those based on microspheres / nanoparticles. Also, particles of this nature are available in several colors. These colors are tagged to different detector species during the method and are differentially detected. i.e. useful for producing colorimetric signals in a "multiplex" during detection reactions. It could be.

[0070] Fluorometric detection involves the generation of a fluorescent signal that, under appropriate excitation stimuli, leads to the subsequent detection of a detector species. This can be achieved through the use of any dye that emits a signal. For example, direct fluorescence detection Dyes available include, but are not limited to: quantum dots, ALEXA dyes, fluorescein, ATTO dyes These include rhodamine, rhodamine, and Texas Red. Method embodiments utilizing fluorescent dye moieties also include those that utilize fluorescence resonance energy transfer (FRET). , for example, based on the use of TaqMan quantitative PCR or molecular beacon-based strategies for nucleic acid detection. Detection can also be performed by increasing or decreasing the concentration of the detector species after binding of the dye. Typically, when using a fluorometric approach, several different detection devices are used. , for example, a CCD camera, a fluorescence scanner, a fluorescence-based microplate reader, or a fluorescence microscope. A microscope or the like can be used to record the production of a fluorescent signal.

[0071] In a further embodiment, a method for detecting the first oligonucleotide probe is provided. The moiety provides a detectable signal, e.g., a colorimetric or fluorometric signal, after contact with the substrate. Those skilled in the art will recognize that several enzyme substrate systems, such as ELISA, It is clear that immunohistochemical detection is available and routinely used in the diagnostic field. Horseradish peroxidase (HRP) is one example. An enzyme conjugated to a first oligonucleotide probe for detection of a detector species in the The use of Provides improved control of signal color development through separate steps. Examples include: glycosyl hydrolases, peptidases, amylases, esterases (e.g., carboxyesterases, glycosidases (e.g., galactosidases), and phosphatases. This list may be used by anyone. The law should not be considered limiting.

[0072] In another approach, the presence of the detector species in step c) can be electrically, e.g. Impedance change or conductometric signal change, amperometric signal change, voltammetry The presence of the detector species is detected by a signal change or a potentiometric signal change. Thus, in one embodiment, the detector species is detected by a change in an electrical signal. The change in the electrical signal is detected by a first oligonucleotide, such as a chemical group that enhances the change in the electrical signal. This may be facilitated by a moiety that allows detection of the oligonucleotide probe. The detection of the signal is so sensitive that the detection portion is simply an oligonucleotide sequence. In some embodiments, the signal is an electrochemical signal, such as a metal, for example gold and carbon. This is enhanced by the presence of chemical groups known to enhance the optical signal.

[0073] In one embodiment, the change in the electrical signal due to accumulation of the detector species is In other embodiments, detection of an electrical signal can be used to detect the presence of an aqueous reaction. A specific site mediated by a second oligonucleotide probe for detection, e.g. This is facilitated by localization of the detector species to the surface of the gas-chemical probe.

[0074] Detector species that are customarily used to detect nucleic acids and are also used in this method. Other techniques that can be used include mass spectrometry (e.g., MALDI or LC-TOF), emission spectroscopy or spectroscopic analysis. , fluorescence spectroscopy or spectroscopy, liquid chromatography, and fluorescence polarization.

[0075] In one embodiment, in step c), carbon or gold, preferably carbon, is used to A colorimetric or electrochemical signal is produced.

[0076] In one embodiment, the detector species is detected by nucleic acid lateral flow. is separated from the other reaction components by its diffusion through a membrane, typically made of nitrocellulose. The nucleic acid lateral flow technique is a rapid and low-cost detection method that can be used for colorimetric, fluorometric, and Various signal readouts can be combined, including nucleic acid readings, and electrical signals. Ralph Flow is well suited for use in detecting the detector species in this method, In one embodiment, the first oligonucleotide in the detector species A leutide probe is used to bind colorimetric or fluorometric dyes, and a detector type is used. The dye is then attached to a lateral flow strip using a second oligonucleotide probe in In this way, nucleic acid lateral flow detection is performed, localizing the nucleic acid to a defined location on the sample. Thus, rapid detection can be performed and the results can be visualized by eye or by a reader. In acid lateral flow, an antibody was used as the detector moiety of the second oligonucleotide probe. The relevant antibodies can be immobilized on a lateral flow strip using a marker. Alternatively, the method may include using a predictor species or detector on a lateral flow strip. Sequence-specific detection via hybridization of one species can be easily performed, A simplified, low-cost alternative to antibody-based assays with improved multiplexing potential This method provides an alternative to the previously known method that does not utilize the two oligonucleotide probes of the present invention. Methods such as SDA typically utilize sequence-specific hybridization-based detection. In contrast, in the present method, the detector species is a position-specific By using selective hybridization-based detection, Carbon or gold nanoparticles are readily available for nucleic acid lateral flow. Depending on the localization of the detector species, carbon or gold will be concentrated locally, resulting in a black or red color, respectively. In one embodiment, the first oligonucleotide probe is a sequence-specific hybridoma. The dye is allowed to bind to the colorimetric dye prior to localization on the strip by hybridization. The antibody contains a moiety, such as biotin, that acts as a stimulatory agent.

[0077] The spatial arrangement of detector species can be determined, for example, by hybridization of detector species at specific locations. If fusion-based binding is permitted, the technique used to detect the detector species and the density In addition to facilitating rapid and specific detection, such physical associations This may enhance the use of the method in multiplex detection of multiple different target nucleic acids. In embodiments, the second oligonucleotide probe is a nucleic acid lateral flow strip or is attached to the surface of an electrochemical probe, a 96-well plate, a bead, or an array surface. Thus, at least one species in the amplification product will have the shape of a detector species at such a position. The second oligonucleotide probe is then localized to a physical location that is easily detected after synthesis. Alternatively, the single-stranded oligonucleotide can be attached to a second polymer that allows its attachment to the solid material. It may be advantageous to use the nucleotide sequence as a moiety to bind to an oligonucleotide probe of the present invention. In this way, the sequence of the oligonucleotide bound to the solid phase can be determined independently of the target nucleic acid sequence. Thus, in one embodiment, the second o The portion of the oligonucleotide probe that allows it to bind to a solid material is a single-stranded oligonucleotide. The single stranded oligonucleotide has high hybridization affinity and It is possible to design methods to improve the efficiency and enhance the performance of the method. For example, In one embodiment of the method, the second oligonucleotide probe is Instead of being directly attached to the trip, it is present in a second oligonucleotide probe. On the strip, which allows efficient hybridization with single-stranded oligonucleotide moieties Separate oligonucleotides with sequences optimized for hybridization are employed. It has been done.

[0078] In various studies, the inventors have synthesized a single-stranded oligonucleotide by ligating it to a second oligonucleotide. The use of nucleic acid lateral flow assays as the binding moiety for the tide probe significantly improved the performance of the assay. For example, GC-rich sequences were hybridized on the strip. Can be used for hybridization on triplicate or for the addition of a second oligonucleotide A longer sequence with a higher Tm can be used to compensate for the length of the probe. The single-stranded oligonucleotide portion may contain one or more modified bases to enhance its affinity or can include an internucleotide bond, such as a PNA, an LNA, or a G-clamp. By using a repeat sequence motif in the single-stranded oligonucleotide portion, the predicted Tm It was found that an unexpected increase in hybridization efficiency was observed when Thus, in one embodiment, the sequence of the single-stranded oligonucleotide portion is a DNA sequence of 2 to 4 bases. A sequence motif. In various studies, the inventors have demonstrated that the detection sensitivity of nucleic acid lateral flow is often 100-fold higher. Significant signal enhancement was observed through the above.

[0079] Thus, in one embodiment in which the presence of the detector species is detected by nucleic acid lateral flow, The nucleic acid lateral flow is a solid material of the second oligonucleotide probe. One or more nucleic acids capable of sequence-specific hybridization with a moiety that allows for the binding of Use acid.

[0080] A further advantage is the need to separate the target nucleic acid sequence from the solid material for binding or from the detection means. This is done by attaching a single stranded oligonucleotide to a first oligonucleotide. As a detection moiety in the oligonucleotide probe and / or as a binding moiety for a second oligonucleotide probe In this way, the relevant solid material for bonding can be used as or a device comprising said solid material, e.g. a nucleic acid lateral flow strip, and / or detection The means can be optimized and defined without taking into account the sequence of the target nucleic acid to be detected. Such "universal" detection devices would need to be modified for each application and target. For example, efficient on-strip hybridization can be achieved without the need for a strip. Oligonucleotide sequences that have the ability to interrogate and not define unintended crosstalk A line corresponding to a matching set of the target nucleic acid sequences is printed, and a method for detecting multiple target nucleic acid sequences is provided. The oligonucleotide primers and probes were developed independently and optimized to efficiently Nucleic acid lateral flow strips being manufactured.

[0081] In some embodiments, the detection can be quantitative. The level of single-stranded target nucleic acid can be quantified in step c). Instead of measuring the end point of a reaction, the reaction may be measured at multiple points in the reaction time series, e.g., colorimetrically, fluorometrically, or This can be achieved by measuring the detector species electrically. Alternative strategies for quantification include In addition, there is a serial dilution of the sample similar to droplet digital PCR. In this manner, the level of single-stranded target nucleic acid in a sample can be determined semi-quantitatively. For example, The intensity of the colorimetric signal on the nucleic acid lateral flow strip indicates the amount of single-stranded target nucleic acid in the sample. Alternatively, an inhibitor can be used, which inhibits To overcome the toxic agent and produce a detectable copy number of the detector species, the single-stranded nucleic acid target The target copy number must exceed a certain defined copy number.

[0082] In the method of the invention, the second oligonucleotide probe is attached to the solid material or In an embodiment, the compound is optionally linked to one or more other moieties that allow for binding to the compound. The oligonucleotide primers and probes can be attached to the solid material or to the solid material. Those skilled in the art will appreciate that the solid-state materials and It will be apparent that the coupling can be achieved in a variety of different ways. For example, , which are useful for the purpose of binding or reacting with appropriately modified oligonucleotide probes. Such a compound has a sufficient density of functional groups, or can bind to them, or Several different solid materials are available that can be functionalized. A wide variety of samples, including beads, resins, surface-coated plates, slides, and capillaries. Such solid materials are available in a variety of shapes, sizes, and forms. Examples of such solid materials used for covalent attachment of include, but are not limited to, glass slides, glass plates, Lass beads, ferrite core polymer coated magnetic microbeads, silica microbeads particles or magnetic silica microparticles, silica-based capillary microtubes, 3D reactions Polymer slides, microplate wells, polystyrene beads, poly(lactic acid) (PL A) Particles, poly(methyl methacrylate) (PMMA) microparticles, controlled pore glass Resins, graphene oxide surfaces, and functionalized agarose or polyacrylamide surfaces Polymers such as polyacrylamide are made by mixing the monomers ( For example, during the polymerization reaction between acrylamide monomers, functionalized oligonucleotides The functionalized oligonucleotide has the additional advantage that it can be covalently bonded to the to produce a solid polymer containing covalently attached oligonucleotides. The polymerization controls the size, shape, and morphology of the resulting oligonucleotide-bound solid material. This provides a highly efficient means of attaching oligonucleotides to solid materials.

[0083] Typically, to bind an oligonucleotide probe to any such solid material, In addition, an oligonucleotide having a functional group at the 3' or 5' end is synthesized. During the nucleotide production process, additions can be made at almost any base position. In this case, specific reactions are performed between functional groups in the oligonucleotide and functional groups on the relevant solid-state material. to form a stable covalent bond to obtain an oligonucleotide bound to the solid material. Typically, such oligonucleotides are linked to a solid material at the 5' or 3' end. For example, two commonly used and reliable conjugation chemistries are thiol (S H) or amine (NH3) groups and functional groups in the oligonucleotide. It can react with a maleimide moiety on the solid support to form a thioester bond, while the amine teeth Reacts with succinimidyl ester (NHS ester) modified carboxylic acids to form amide bonds Several other chemistries can also be used. For use in carrying out the method, as well as chemical conjugates with solid materials, It is possible and potentially useful to synthesize oligonucleotide probes directly on solid-state materials. It is beneficial.

[0084] In other embodiments, the second oligonucleotide probe binds to the solid material. One strategy utilizes affinity binding methods. and thereby attaching a moiety that allows for specific binding to the oligonucleotide probe. This can facilitate binding to its associated affinity ligand, e.g., an antibody. Using an affinity tag, such as an antigen-binding or polyhistidine tag, or complementary Nucleic acids are bound to solid materials, such as nitrocellulose nucleic acid lateral flow strips. This can be accomplished using acid-based hybridization. A typical such moiety is biotin, which can itself be attached to a bead or another solid surface. Binds with high affinity to streptavidin or avidin bound to a surface can be done.

[0085] The presence of two or more different target nucleic acids of defined sequence can be detected in the same sample. In one embodiment of this method, a different oligonucleotide is prepared for each of two or more target nucleic acids. Using oligonucleotide primers and oligonucleotide probes, a series of steps a) and b) are separately performed. a), b), and c), where the separate steps can be performed simultaneously. In an embodiment, a set of oligonucleotide primers and oligonucleotide probes The probes are used to detect one target nucleic acid in a sample and another set of oligonucleotide probes are used to detect the target nucleic acid in a sample. The primers and oligonucleotide probes are used to detect other target nucleic acids in the same sample. Detection of detector species produced from two or more different primer / probe sets is , each with a specific signal, e.g., a different colorimetric dye or can be conjugated to a fluorometric dye or enzyme. Alternatively, multiplex detection can be performed and attaching a second oligonucleotide directly or indirectly through a moiety that allows for binding to the solid material. This can be achieved by binding the leutidine probe to a solid material. Rather than relying on different detection means, such an approach would involve a series of different steps a), b), and c). Thus, for example, the use of a nucleic acid lateral separation technique is used to separate the detector species produced by A single dye can be used to detect multiple different target nucleic acids in a single flow-through system, Each different detector type identified corresponds to a specific printed line on the lateral flow strip. The nucleic acid sequence is localized in the nuclease domain and is subjected to direct or indirect sequence-based hybridization with a second oligonucleotide probe. Hybridization forms the basis for differential detection. A detection array can be used, in which a plurality of different second oligonucleotide probes are arrayed. This allows multiplexing reactions to be performed using multiple different deoxyribonucleic acid (DTA) antibodies. Detector species are produced simultaneously, and each detector species allows for multiplex detection. It is localized by hybridization to discrete regions of the array.

[0086] The above detection processes, such as nucleic acid lateral flow and electrical detection, and Its ability to readily detect multiple different target nucleic acids is due to the fact that the method uses two oligonucleotides This is achieved by essentially requiring a probe. Therefore, the detection process The advantages of the method of the present invention over known methods are effectively demonstrated.

[0087] The present invention is applicable to a variety of fields and applications requiring the detection of a target nucleic acid of defined sequence in a sample. The present invention has broad utility for determining the presence of a target nucleic acid sequence in a sample quickly, inexpensively, and Through a non-limiting list of applications, the inventors They believe that the invention has applications in diagnostics, forensics, agriculture, animal health, the environment, defense, human genetic testing, prenatal testing, and other areas. Laboratory testing, blood contamination screening, pharmacogenomics or pharmacokinetics, and microbiology, clinical and biomedical research. is a biological sample, such as a human sample. The sample may be a human sample, a forensic sample, an agricultural sample, It may be a veterinary sample, an environmental sample, or a biodefense sample.

[0088] Detection of target nucleic acids can be used to detect and treat a disease or condition, such as, but not limited to, HIV, influenza, and the like. Influenza, RSV, Rhinovirus, Norovirus, Tuberculosis, HPV, Meningitis, Hepatitis, MRSA, Mullet, Clostridium difficile, Epstein-Barr virus, malaria, plague , polio, chlamydia, herpes, gonorrhea, measles, mumps, rubella, cholera, or infectious diseases including, but not limited to, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, Cancer, prostate cancer, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma For the diagnosis, prognosis, or monitoring of cancer, including human genetic testing, prenatal testing It can be used in the fields of drug discovery, blood contamination screening, pharmacogenetics, or pharmacokinetics. do.

[0089] The present invention relates to, for example: nasal swabs or aspirates, nasopharyngeal swabs or aspirates, throat swabs or aspirates. fluid, buccal swab or aspirate, blood or blood-derived sample, urine or urine-derived sample, sputum or sputum-derived sample Samples, feces or feces-derived samples, cerebrospinal fluid (CSF) or CSF-derived samples, and gastric juice or gastric juice-derived samples. It can be used with a wide variety of samples, including human or animal samples derived from any form of tissue biopsy or body fluids. The inventors also used the following clinical specimens: nasal swabs for VTM, nasopharyngeal swabs for VTM, Swabs, thin prep medium, throat swabs in liquid Amies, HSV sore swabs in M4 medium, synovial fluid, 2 M NaOH / isopropanol, followed by sputum treated with DNA capture beads, rectal swabs in TE; Fecal samples, homogenized and treated with DNA capture beads, CSF, APTIMA swabs, sheep Water, oral swabs in liquid Amies, urine, VRE swabs in TE, pleural fluid, whole blood, K2EDTA plasma, L. hepatica The method has been performed on a wide range of samples, including plasma, blood serum, and at least 10-20% phosphorus. These experiments demonstrate the remarkable versatility and relevance of this method for a variety of clinical applications. This demonstrates that no inhibition was observed in the relevant samples, which is consistent with the heparin inhibitor that inhibits PCR. and other methods that are inhibited by inhibitors found in biological samples, such as phytic acid. In stark contrast, the method is low-cost and easy to use without requiring any complicated sample preparation procedures. The potential of using this method in disposable equipment is demonstrated.

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

[0091] The single-stranded target nucleic acid of this method may be naturally occurring or non-naturally occurring. The acid may be generated in situ or produced from naturally occurring nucleic acids prior to carrying out the method. The single-stranded target nucleic acid of this method may be subjected to one or more additional steps carried out prior to or simultaneously with step a). The additional steps may include one or more additional enzymes such as a polymerase and a restriction enzyme. The above enzymes can be included in the production of target nucleic acid in this manner. Allow for multiplex assays and / or overcome background from 1 There are several potential advantages to this, for example, without amplifying the It is possible to carry out highly specific conversion of nucleic acid material in a biological sample. The sample may be, for example, It is processed, purified, subjected to buffer exchange, subjected to exome capture, and partially freed from contaminating materials. and / or can be converted to a single-stranded target nucleic acid for methods involving one or more modified dNTPs. The "true" target nucleic acid to be detected in the sample is converted with high confidence (<1:1, 1:1 or may be 1:>1, i.e., may have several amplification elements) For the implementation of the method, detection of the "surrogate" target nucleic acid is This would allow for the detection and / or quantification of "true" nucleic acids. The production of surrogate targets derived from natural targets can thus be used to generate method variants having any desired sequence. The target nucleic acid can be specifically generated. Single-stranded target nucleic acid can be generated, for example, by strand invasion. In one embodiment, two complementary single-stranded nucleic acids are obtained from double-stranded DNA after dissociation of the two strands. The target is present and is reacted with the same oligonucleotide primer and probe in a reciprocal process. If the target is the genome of a -ve single-stranded RNA virus, the target can be amplified and detected in the sample. In some cases, +ve transcripts may also be present, and the same oligonucleotide primers and probes may be used. In this method, either or both strands are amplified as single-stranded target nucleic acid, And it can be detected.

[0092] The present invention also relates to cell-free DNA and epigenetic modifications, such as CpG methylation, of DNA sequences. It is anticipated that this method will be useful in screening samples for specific cancers. Such epigenetic modifications of relevant target genes may be useful biomarkers in many diseases and pathological conditions. The importance of epigenetic modifications in human diseases is currently being evaluated. Considering the increasing demand for DNA polymerases and / or restriction enzymes, Specific assessment of epigenetic modifications of specific target nucleic acid biomarkers based on differentiated activity Thus, in one embodiment, the present invention can be used to detect a target nucleic acid The gene may contain epigenetic modification sites such as methylation sites. Alternatively, the gene may be a "substitute" for carrying out the above-mentioned method. The "true" nucleic acid used to produce the "controlled" target nucleic acid includes the epigenetic modification site.

[0093] A further aspect of the invention relates to a kit for use in detecting nucleic acids of defined sequence in a sample. Therefore, the present invention also provides the following: a) a first oligonucleotide primer and a second oligonucleotide primer; Thus, the first primer has a restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction, and A single strand of a defined sequence hybridizes to a first hybridization sequence in a target nucleic acid. and the second primer comprises a region capable of cleaving a restriction enzyme recognition region in the 5' to 3' direction. A sequence and cleavage site and a second sequence upstream of the first hybridization sequence in the target nucleic acid. A region capable of hybridizing with the reverse complement of the hybridization sequence of , the first oligonucleotide primer and a second oligonucleotide primer; b) a nicking enzyme that recognizes the recognition sequence of the first primer and forms the cleavage site The second primer is not a first restriction enzyme and a nicking enzyme that can cut the a second restriction enzyme capable of recognizing said recognition sequence and cleaving said cleavage site; c) strand-displacing DNA polymerase; d) dNTPs; e) one or more modified dNTPs; f) the first single strand of at least one species of amplification product produced in the presence of said target nucleic acid It is capable of hybridizing to a detection sequence and is linked to a moiety that allows its detection. , a first oligonucleotide probe; and g) a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product; A single stranded detection sequence is hybridizable to the solid material or to the solid material. and a second oligonucleotide probe coupled to an enabling moiety. do.

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

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

[0096] In another embodiment, the first oligonucleotide probe of the kit comprises the first and and some complementarity with the hybridizing region of one of the second oligonucleotide primers; For example, the second oligonucleotide probe of the kit has a complementarity of 5 or more bases. The probe is a reverse of the hybridizing region of the other of the first and second oligonucleotide primers. It has some complementarity with the complementary strand, for example, 5 or more bases.

[0097] In a further embodiment, the first and / or second oligonucleotide probes are The lobes are more or less spaced apart from the gap between the first and second hybridization sequences of the target nucleic acid. It may have a complementary or reverse complementary nature.

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

[0099] The kit comprises a detector for detecting the presence of a detector species produced in the presence of said target nucleic acid. For example, the kit may further include a nucleic acid lateral flow strip, an electrode, Chemical probes, 96-well plates, beads or array surfaces, and / or colorimetric dyes or fluorometric dyes, and / or devices for detecting changes in electrical signals, and / or It may include carbon or gold.

[0100] In various embodiments, the target nucleic acid and kit components included in the kit, e.g. the first oligonucleotide primer in at least one species of the amplification product; and and / or the second oligonucleotide primer, and / or the first restriction enzyme, and / or or said second restriction enzyme, and / or said DNA polymerase, and / or said dNTPs, and / or the one or more modified dNTPs, and / or the first oligonucleotide probe, and / or The second oligonucleotide probe and / or the first or second single-stranded detection sequence is As defined herein for the methods of the invention. For example, the kit may include Any combination of the features of the components described herein may include, but is not limited to, the following: One of the first and second oligonucleotide probes is elongated by a DNA polymerase. blocked at the 3' end and optionally containing one or more sequence mismatches and / or one or more modifications, e.g., phospho can be cleaved by the first or second restriction enzyme due to the presence of a dithioate bond. said first restriction enzyme and said second restriction enzyme are the same restriction enzyme; said one or more The modified dNTP is an α-thiol modified dNTP; allowing detection of the first oligonucleotide probe. The part that can be used is a colorimetric or fluorescent dye, or a colorimetric or fluorescent dye. a moiety that can be attached to a nucleic acid, such as biotin; The portion of the lobe that allows binding to a solid material is optionally selected from three or more of the DNA sequence motifs of 2 to 4 bases. a single stranded oligonucleotide comprising repeated copies of The primer is a primer that is upstream of the restriction enzyme recognition sequence and the cleavage site, for example, at the 5' end and and a stabilizing sequence, for example, 5 or 6 bases in length; the hybridizing region of the target nucleic acid is 6 to 30 bases long, for example 9 to 16 bases long; The first and second hybridization sequences are separated by 0 to 15 or 0 to 6 bases, and in some embodiments In an embodiment, they are separated by 3 to 15 or 3 to 6 bases, such as 5, 7, or 11 bases, Or they may overlap by, for example, 1-2 bases.

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

[0102] The kit also includes other additives such as reaction buffers, salts, e.g., divalent metal ions, additives, and excipients. Reagents may also be included.

[0103] Kits according to the invention may be provided with instructions for carrying out the methods according to the invention. stomach.

[0104] The present invention also provides a method for detecting single-stranded target nucleic acids of defined sequence in a sample, comprising the steps of: The use of the kit is also provided.

[0105] Any optional and / or preferred embodiment of the invention described herein relating to the method of the invention The aspects also apply in relation to the kit of the invention and its uses, and vice versa. I want you to understand this.

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

[0107] The method of the present invention also provides a method for amplifying a nucleic acid signal derived from a target nucleic acid of defined sequence. Such a method can be used independently of the detection step c) for detecting amplified The resulting signal can then be used to detect the target nucleic acid at a future time and / or at an alternative location, if desired. It can be used if the amplified The signal produced may be a predictor or detector species produced during the practice of the method. Thus, in a further embodiment, the present invention provides a method for detecting a target of a defined sequence in a sample. A method for amplifying a nucleic acid signal derived from a nucleic acid, comprising steps a) and all or part of the method of the present invention. comprises part, for example part i. or ii., of step b).

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

[0109] In connection with the method of the present invention for detecting the presence of a target nucleic acid of a defined sequence in a sample, All optional and / or preferred embodiments of the invention described herein may be used in combination with the defined sequence targets. It is understood that the present invention applies in conjunction with methods for amplifying a nucleic acid signal derived from a target nucleic acid. I want to.

[0110] The following examples further demonstrate various aspects and embodiments of the methods described herein. These examples are not to be construed as limiting in any manner. It won't happen. EXAMPLES

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

[0112] Nucleic acid lateral flow: Carbon nanoparticles are tethered to various biotin-binding proteins, e.g. It is conjugated to streptavidin via non-covalent adsorption. A carbon suspension was prepared in borate buffer and then ultrasonicated using a probe sonicator. The carbon was then incubated at room temperature to remove the biotin. Carbon was either used directly in the reaction mixture or attached to a glass fiber. Conjugate pads were applied. Lateral flow strips were prepared according to the manufacturer's instructions (Merck Millipore, The conjugate pads containing lyophilized sugars and The conjugate pad, which contains an additive used to improve the visual appearance, is then placed over the sample pad. The lateral filter was constructed by combining a nitrocellulose membrane and an adsorption pad. Prior to using it in a low strip, the reverse phase of the sequence to be detected in this method is Related oligonucleotides containing complementary strands are printed at defined positions on a nitrocellulose membrane. and bonded to the membrane via UV crosslinking.

[0113] Example 1 (The second oligonucleotide probe was attached to a solid material, nitrocellulose lateral freeze-thaw. (Method of bonding with lip) In this example, the second oligonucleotide probe is attached to a solid material, nitrocellulose lamination. The first oligonucleotide probe is bound to the teraflow strip and the amplification step a) The method is performed without contacting the sample at the same time.

[0114] In the 5' to 3' direction: 7 base stabilization region; 5 base non-nicking restriction enzyme recognition and a 12-base sequence comprising the reverse complement of the first hybridization sequence in the target nucleic acid. A first oligonucleotide primer having a total length of 24 bases and including a hybridization region of A second hybrid was designed that contained the same stabilizing region and restriction enzyme recognition sequence, but with a second hybrid in the target nucleic acid. A 12-base hybridization sequence capable of hybridizing with the reverse complement of the DNA fragment A second oligonucleotide primer was designed containing the size region. The first and second restriction enzymes are the same. The restriction enzyme has a 5-base recognition sequence The target nucleic acid is an asymmetric double-stranded cleavage restriction enzyme having a top strand cleavage site downstream of the The first and second hybridization sequences are separated by one base.

[0115] The oligonucleotide primers are designed using the target nucleic acid, thereby The nucleotide base downstream of the cleavage site on the reverse complementary strand becomes adenosine, resulting in an α-thiol dATP is utilized as the modified dNTP in this method. It is inserted by a polymerase to prevent cleavage of the reverse complementary strand.

[0116] In the 5' to 3' direction: a 12-base region complementary to at least one species in the amplification product; the spacer region between the first and second nucleotides; and the 3' biotin modification added during synthesis. A first oligonucleotide probe is designed, wherein the biotin modification is It is possible to combine the leutidine probe with colorimetric dyes and carbon nanoparticles. Carbon adsorbed with thion-binding protein was prepared and the first oligonucleotide probe was In the 5' to 3' direction: an intermediate spacer containing 10 thymidine bases; a second single-stranded detection sequence downstream of the first single-stranded detection sequence in said at least one species in A second oligonucleotide having a total length of 49 bases and including a 3× repeat sequence of a 13 base region capable of hybridizing with Approximately 30 pmol of the second oligonucleotide probe was used. The probes were printed onto nucleic acid flow strips.

[0117] 1.6 pmol of first primer; 0.1 pmol of second primer; Enzo Life Sciences 250 μM 2′-deoxyadenosine-5′-O-(1-thiotriphosphate) SP-isomer (Sp-dATP-α-S); 60 μM each of dTTP, dCTP, and dGTP; 2 U of restriction enzyme; and 2 U of Bacillus strand-displacing DNA polymerase. A reaction mixture containing the ribosomal enzyme was prepared. For nucleic acid targets (single-stranded DNA targets), a suitable reaction buffer was used. A total reaction volume of 10 μl was used to assay various levels of target (++=1 amol, +=10 zmol, NTC=no target). The reaction mixture was incubated at 45°C for 7 or 10 minutes. μl of termination reaction mix at 0.056 mg / ml -1 60 μl of lattice containing 100 μl of conjugated carbon After addition of the lateral flow running buffer, the nucleic acid was applied to the printed lines on the lateral flow strips. A second oligonucleotide probe bound to the strip was then loaded.

[0118] Figure 5 shows a photograph of a lateral flow strip obtained in the practice of this example. The arrows indicate A second oligonucleotide probe was printed onto the nitrocellulose strip, thus The positions where positive signals appear are shown. Clear black areas correspond to the presence of carbon signals. A dark line was only observed in the presence of target nucleic acid at both target levels and at both time points, demonstrating the method of the present invention. The method demonstrates rapid and sensitive detection of target nucleic acid sequences.

[0119] Example 2 (The first oligonucleotide probe inhibits the extension by the DNA polymerase at the 3' end. and the probe is blocked and cannot be cleaved by the first or second restriction enzyme, and the probe is (Contacting the sample, carrying out the method) In this embodiment, the first oligonucleotide probe is elongated by the DNA polymerase. The length of the probe is blocked at the 3' end, and the probe cannot be cleaved by the first or second restriction enzyme. This demonstrates the implementation of an embodiment of the method in which step a) is performed while simultaneously contacting the sample. In an embodiment, the inventors did not observe any significant inhibition of the amplification rate and found optimal cyclic amplification. The predictor species were accumulated in real time without interrupting the width process. The inventors did not observe any inhibitory effect on the amplification process in the above embodiment. Not only did we obtain a 100-fold increase in the amount of detector species. An unexpected enhancement of the generated signal was observed.

[0120] (Example 2.1) The first oligonucleotide probe is elongated by the DNA polymerase is blocked at the 3' end and cannot be cleaved by the first or second restriction enzyme, The embodiment of the method used in Example 1 was used in which the sample was contacted at the same time as step a) was performed. A variation of the assay was designed. The same oligonucleotide primers as those used in Example 1 were used. -, restriction enzymes, dNTPs, modified dNTPs, and polymerase were used, in the 5' to 3' direction: 5' Biotin modification; 8-base middle region; hybridizes to at least one species in the amplification product 13-base region that can be used for the first oligonucleotide; 3' phosphate modification (biotin modification) The probe can be combined with colorimetric dyes and carbon nanoparticles. The first oligonucleotide, which is an alternative to the first oligonucleotide, is 21 bases long and contains a substitution type DNA polymerase that prevents its extension. We designed a nucleotide probe to detect the binding of biotin to a protein. The DNA was prepared and saturated with the first oligonucleotide probe.

[0121] In the 5' to 3' direction: a first single-stranded detection sequence in said at least one species in the amplification product a 14-base region upstream of the second single-stranded detection sequence that can hybridize to the a spacer sequence; a repeat of a 14-base hybridizing region; a second 6-base intermediate spacer sequence; and A second oligonucleotide probe with a total length of 51 bases containing a 10× thymidine-based spacer was used. Approximately 30 pmol of the second oligonucleotide probe was added to the nucleic acid flow cytometer. Printed on the strip.

[0122] 0.8 pmol of first primer; 0.8 pmol of second primer; 0.6 pmol of first oligonucleotide Nucleotide probe; 300 μM Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 2 U A reaction mixture was prepared containing 10 U of restriction enzyme; and 2 U of Bacillus strand-displacing DNA polymerase. Acid targets (single-stranded DNA targets) were incubated at various levels in a total reaction volume of 10 μl in the appropriate reaction buffer. (++=1 amol, +=10 zmol, NTC=no target control). The reaction was incubated at 45°C for 6 min. Then, 5 μl of the termination mix was added to the 0.03 mg / ml -1 Conjugates of After adding 60 μl of lateral flow running buffer containing the carbon, the nucleic acid lateral flow strip was If the first oligonucleotide probe was not present in the reaction, Control reactions were performed to demonstrate that no detector species were produced. - To control for any unintended effects due to the presence of the probe during the detection of the strip To achieve this, an equivalent level (0.6 pmol) of probe was added to the control after step a).

[0123] FIG. 6A presents a photograph of the nucleic acid lateral flow strip after its color development. When oligonucleotide probes were provided, the deposition of carbon nanoparticles was clearly A clear signal was observed at both target levels. As expected, the first oligonucleotide was When no tid was provided, no signal was detected at either target level. In an embodiment, the first oligonucleotide probe is elongated by the DNA polymerase. The probe is blocked at the 3' end and cannot be cleaved by the first or second restriction enzyme, In an embodiment of the method, the detector species is contacted with the sample simultaneously with carrying out a). In contrast to Example 1, the use of equal concentrations of first and second o- It is noteworthy that the present invention provides oligonucleotide primers that allow for more rapid amplification. do.

[0124] (Example 2.2) Next, we demonstrate the versatility of the above embodiment of the method using completely different target nucleic acids. Separate assays were designed to detect the oligonucleotide primers and The probes were designed for the relevant target nucleic acid, a single-stranded DNA target, as described in Examples 1 and 2.1. Calculated.

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

[0126] FIG. 6B shows a photograph of the nucleic acid lateral flow strip after its development. Under these conditions, a clear positive signal was visible, and no signal was visible in the no target control. This allows for proper design of the assay and the first oligonucleotide probe. The extension by the DNA polymerase is blocked at the 3' end, and the first or second restriction enzyme and the probe is contacted with the sample simultaneously with carrying out step a). The robust potential of the method was demonstrated. As expected, only minimal signals were observed in the control assay, indicating that the detector species In order to efficiently produce the first oligonucleotide primer, the amplification step in step a) is carried out simultaneously. It has been demonstrated that proper hybridization of the lobes is required.

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

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

[0129] 0.032 mg / ml -1 65 μl of the appropriate buffer solution containing carbon adsorbed with biotin-binding protein 0.5 pmol of target A and first oligonucleotide probe for target B in buffer; 0.5 pmol of target A and Reactions were constructed containing A, B, C, D, and a second oligonucleotide primer for A. Add target (+=0.1 pmol, ++=1 pmol) to separate reactions and add both targets together. A no target control (NTC) was also performed.

[0130] Figure 7A shows a photograph of the lateral flow strip obtained in this experiment. Distinct black spots corresponding to carbon deposits were observed at both target levels and in both uptake Furthermore, when both reactions were performed simultaneously, both targets A and B were No significant difference in background signal or between the different assays was observed. No crosstalk was observed.

[0131] To demonstrate the robustness of the method, further experiments were carried out to identify three copies of the defined sequence in the sample. To demonstrate the potential of the method for detection of different target nucleic acids, three separate assays were performed. The same methodology as above was used. Figure 7B shows the resulting lateral flow strip. Photographs of the target P1, P2, and P3 are shown individually and in various combinations as shown. The single-stranded oligonucleotide detection portion of the second oligonucleotide probe The reverse complement to was printed in a separate line on the nucleic acid lateral flow strip. In all cases, the signals were localized to carbon and fluorescein at the predicted positions for rapid and sensitive detection. The results show that the accumulation of bound detector species is consistent with no unintended crosstalk between assays. In a comparable experiment involving four separate assays, The potential of the method for detection of four different target nucleic acids (P1, P2, P3, and P4) of defined sequences was demonstrated. The results are shown in Figure 7C. In this four-target experiment, P4 was used as a positive control and was The lateral flow diagram shown shows that the 100-fold increase in the chromatin was due to the addition of the 100-fold increase in the chromatin. The photograph of the strip shows the predicted presence of the relevant detector species bound to the carbon. Such a multiplex assay shows a clear black band at the selected position. The potential of the method to be used for diagnostic testing of diseases caused by several different pathogens. Here, detecting the presence of the detector species in the control assay demonstrates that the method Demonstrated successful implementation and one or more other detectors on the lateral flow strip Visualization of one or more pathogens indicates the presence of the relevant causative pathogen in the appropriate clinical specimen. Observing the presence of multiple infections in the same specimen is of great value in such diagnostic applications, e.g. Although rare in the field, the method of the present invention detects multiplex reactions In Figure 7D, four targets (P1, P2 The results of an experiment in which different combinations of P1, P2, and P3 were added were shown. Each target was detected separately. , each target could be omitted without producing nonspecific background, whereas the other three targets The ability to detect demonstrates the remarkable specificity of detection of the methods of the invention.

[0132] In the above and various other experiments, we also demonstrated that the β-glucosidase inhibitor ... Multiplexing for detection of 3-5 targets at 17 ymol (600 copies) or 17 ymol (10 copies). In this example, the inventors performed a genomic assay in which the samples were enriched in a defined sequence. The potential of the method to detect the presence of two or more different target nucleic acids, e.g., nucleic acid lateral flow This study clearly demonstrated the potential for rapid, low-cost signal detection using a defined sequence. The ease with which two or more different target nucleic acids can be detected in the same sample is an advantage of the method of the present invention. For each additional target to be detected, an additional set of targets is generated. A oligonucleotide primer is required. Additional primers will not form non-specific amplification products. This is because the tendency of the prior art methods not involving temperature cycling is increased. The present invention presents a significant challenge in detecting the presence of two or more different target nucleic acids. In this regard, this issue has been addressed by, for example, the use of modified bases, the selection of improved enzymes, and additional sequence-specific hybridization. Detectors using oligonucleotide probes that exploit hybridization events This is overcome by the enhanced specificity resulting from the formation of specific species.

[0133] Example 4 (The first and second hybridization sequences in the target nucleic acid are separated by 5 bases. (implementation of the method) In this embodiment, the first and second hybridization sequences in the target nucleic acid are 5 The oligonucleotide primers are separated by a base. is absent, and two oligonucleotide primers are hybridized in the first and second If gaps are designed between the sequences, they are only generated in the amplification product in a target-dependent manner. The ability to use target-derived sequences produced may result from synthesis from one or from interprimer binding. The specificity is enhanced in embodiments of the method that can overcome any background signal that may occur. In said embodiment, if the amplification product contains the appropriate target-derived sequence, In order to produce a detector species only if the first or second oligonucleotide is Sequence-specific hybridization of the tide probe was performed in two hybridization regions. The design utilizes the gap between the regions.

[0134] In this example, we identify first and second hybridization sequences in the target nucleic acid. A second oligonucleotide for a variety of different amplification products that differ only in the sequence of the gap between the To demonstrate hybridization of the leucine probe, various assays were designed. The second oligonucleotide probe was a 11-nucleotide probe for at least one species in the amplification product. The first oligonucleotide was designed to contain a base hybridizing region at its 5' end. A 7-base sequence that is the reverse complementary sequence of the nucleotide primer and a gap between the two primers It consists of a 5-base sequence that is the reverse complement of the additional target-derived sequence in the amplified product obtained from The second oligonucleotide probe is composed of 5× thymidine salt in the 5′ to 3′ direction. A 12-base single-stranded oligonucleotide with an intermediate spacer for binding to the solid-state material 30 pmol of an oligonucleotide containing the reverse complementary sequence of the above-mentioned portion was printed. A nitrocellulose nucleic acid flow strip was prepared. The primer has the same sequence as the second oligonucleotide primer, but is modified with a 5' biotin and a 3' Contains phosphate modifications and phosphorothioate internucleotide linkages at the restriction enzyme cleavage sites. It was designed to be

[0135] Four different artificial target nucleic acid sequences (T1, T2, T3, and T4) were designed, each of which has a first and The hybridization sequence of the first and second hybridization sequences was identical to that of the first and second hybridization sequences. There were five base differences between the hybridization sequences: T1 was the second oligonucleotide, Suitable for detection of perfect complementarity to the 11-base hybridizing region of the nucleotide probe T1 contains 4 mismatches out of the 5 bases in the gap; T2 contains 4 mismatches out of the 5 bases in the gap; T3 contains 1 mismatch in the gap The amplification product was designed so that four of the five bases in the sequence were removed, so that the species of the amplification product was four bases. T4 contains two mismatches out of the five bases in the gap.

[0136] 3.6 pmol of first oligonucleotide primer; 1.8 pmol of second oligonucleotide Primers; 2.4 pmol of the first oligonucleotide probe; 300 μM Sp-dATP-α-S, 60 μM dTTP, dCTP, dGTP; 12 U restriction enzyme; 12 U in a total reaction volume of 60 μl in the appropriate reaction buffer Reactions were constructed containing 1 amol of target (T1 After adding T1, T2, T3, or T4), incubate at 45°C for 6.5 minutes, then add 53.5 mL of 60 μl of 1 μl of reaction mixture was run on the lateral flow strip. Prior to application of the reaction mixture, 1.5 pmol of the second oligonucleotide probe and 2 μg of biotin-linked The carbon adsorbed with the conjugate protein was deposited on the conjugate pad and allowed to dry for 5 minutes. Ta.

[0137] Figure 8 shows a photograph of the nucleic acid lateral flow strip obtained in this experiment. The resulting strips are bound to detector species bound to a solid material of nitrocellulose. The resulting figure shows clear black lines corresponding to the carbons that were ligated to the oligonucleotide primers. The assays developed in this example, including the ELISA and probes, functioned properly and were rapid. It has been demonstrated that it has the potential for highly sensitive detection. The reaction did not show any carbon corresponding to a positive signal. Both the match and the removal of four bases were generated during the reaction of the second oligonucleotide. It has been shown that this reduces the ability of the redactor species to hybridize efficiently. A faint signal was observed on the strips produced using T4. A dimer that can bind to a line on a strip with only two mismatched bases. The second successfully hybridized with a predictor species producing a tector species. This shows that the ability of the oligonucleotide probe to react with the polysaccharide is significantly reduced. Acrylamide gel electrophoresis was performed with replicate reactions to determine the total number of replicates for all targets. It was confirmed that the reaction worked properly and produced a significant amount of amplified product. In reactions performed with target T3, the expected size shift was visible.

[0138] In this example, the first and second oligonucleotide probes, which are essential features of the present invention, are Not only does the primer pair provide rapid and sensitive detection of amplification products, but it also Additional target sequences in the amplified products beyond those resulting from hybridization alone We demonstrate how this powerful technique can be used to provide a uniqueness check for the base. , non-target specific binding in a particular assay due to synthesis from 1 or inter-primer binding The method of the present invention overcomes the known problems of the prior art methods arising from background amplification. , showing enhanced specificity compared to prior art methods, whereas highly sensitive detection and rapid, low Demonstrate that visibility into cost results is maintained.

[0139] Example 5 (The portion of the second oligonucleotide probe that allows binding to the solid material is an antigen. , binding the corresponding antibody to a solid surface, a nitrocellulose lateral flow strip; (Implementation of the method) In the method of the present invention, several different portions are bound to the second oligonucleotide probe. It can be used as a moiety for bonding with a solid material. In this embodiment, the second The portion of the oligonucleotide probe that allows it to bind to a solid material is the antigen, and the corresponding The method comprises binding an antibody to a solid surface, a nitrocellulose lateral flow strip. Demonstrate that it can be implemented.

[0140] The second oligonucleotide probe has homology to at least one species in the amplification product. The 32-base sequence including the region that binds the 3' digoxigenin NHS ester modification added during synthesis. The antibody was designed to contain the Fab fragment anti-digoxigenin antibody purified from sheep (Sigma-Ald Rich Co.) was immobilized onto the nucleic acid lateral flow strips by spotting and air drying. Ta.

[0141] The performance of the second oligonucleotide probe was evaluated using a 100-fold increase in the activity of the target at various levels (+++=1 pmol; ++= 0.1 pmol; +=10 fmol; NTC=no target control) at 0.016 mg / ml -1 Biotin-binding proteins The carbon-containing adsorbed carbon is required for detection using the carbon-based nucleic acid lateral flow reaction. This was demonstrated in an experiment where 60 μl of the designed reaction buffer containing the necessary reagents was added. The plate was prepared by centrifugation of 0.5 μg of antidigoxigenin in 0.2 μl of buffer containing 2.5 mM boric acid and 0.5% Tween 20. Genin Fab fragments were spotted onto the strips to prepare the solution. The reaction mixture was incubated at 45°C for 2 min and then dried in a nitrocellulose membrane. After the entire reaction mix for each reaction was aliquoted and the designed detector species was formed, Applied to flow strips.

[0142] Figure 9 shows a photograph of the lateral flow strips produced in this experiment. Black spots corresponding to carbon deposits on the strip were visible at each target level and NTC This indicates that the detector species is specifically detected. Biotin-Based Affinity Interactions for Binding (Carbon) and Solid Materials This example illustrates the combination of antibody-based affinity interactions for binding moieties. , Different approaches available for binding the second oligonucleotide probe to the solid material From this perspective, it serves the purpose of demonstrating the versatility of this method.

[0143] Example 6 (The moiety that enables the second oligonucleotide probe to be bound to a solid material is a triamine. A single stranded oligonucleotide containing four repeat copies of a base DNA sequence motif, (a) carrying out a method in which the reverse complement of a strand oligonucleotide sequence is bound to a solid material; This embodiment allows for the attachment of a second oligonucleotide probe to a solid material. The target region is a single-stranded oligonucleotide that contains four repeated copies of a three-base DNA sequence motif. As described above, the detection portion of the second oligonucleotide probe is An embodiment of the method utilizing single stranded oligonucleotides as nucleic acid probes is by nucleic acid lateral flow. This method facilitates detection of multiple different target nucleic acids in the same sample. We present a simple and versatile embodiment of the method. Furthermore, the single-stranded oligonucleotide detection moiety Predefined and optimized for efficient on-strip hybridization and detection To enhance sensitivity and provide for efficient scale-up manufacturing of nucleic acid lateral flow strips. This can be done.

[0144] In one embodiment of the present invention, the inventors have developed a method for the preparation of a nucleic acid sequence comprising multiple repeated copies of a DNA sequence motif. On-strip hybridization by use of single-stranded oligonucleotide detection moieties In this example, we observed unexpected improvements in the performance of the 100% 15 ... Here we present the results of a second assay directly bonded to the lateral flow strip. The performance of the oligonucleotide assay was evaluated by determining whether the nucleotide sequence of the oligonucleotide was a repeat of four copies of a three-base DNA sequence motif. The detection of the single-stranded oligonucleotide is greatly enhanced by the use of a single-stranded detection moiety containing The reverse complement of the DNA sequence is attached to the lateral flow strip.

[0145] (Example 6.1) The first oligonucleotide probe is elongated by the DNA polymerase is blocked at the 3' end and cannot be cleaved by the first or second restriction enzyme, The assay was designed to utilize an embodiment of the method in which step a) is performed while simultaneously contacting the sample. In the 5' to 3' direction: 5' biotin modification; 7-base middle region; 5 bases that are not nicking enzymes the first restriction enzyme recognition site in the target that contains a phosphorothioate bond at the restriction enzyme cleavage site a 13-base region capable of hybridizing with the hybridization region of The first oligonucleotide probe is modified with a colorimetric dye, cation, by biotin modification. The phosphate modification allows the binding of the phosphate group to the carbon nanoparticles by strand-displacing DNA polymerase. The first oligonucleotide probe was designed to have a total length of 25 bases, including the .

[0146] Two alternative second oligonucleotide probes for detecting the same target species (I and II) The second oligonucleotide probe, "I", was designed to address the following in the 5' to 3' direction: 14 bases capable of hybridizing with the reverse complement of the second hybridization sequence The nucleic acid was designed to contain 3x repeat sequences of the region; and 9x thymidine base spacers. Flow strips were prepared with spots containing 30 pmol of probe.

[0147] In the 5' to 3' direction: hybridizes with the reverse complement of the second hybridization region in the target. a 14-base region that can be ligated; an intermediate spacer of 5× thymidine bases; and a second oligonucleotide. A three-base sequence motif that serves as a moiety enabling the binding of a polynucleotide probe to a solid material. The second oligonucleotide comprises a 12-base single-stranded oligonucleotide portion containing 4x repeats of the nucleotide sequence of The oligonucleotide probe "II" was designed. From 5' to 3': 11× thymidine base spacer 3, which forms a moiety that allows the second oligonucleotide II to be bound to a solid material; Add a 36-base region containing 12x repeats of the reverse complement of the base sequence motif. The second oligonucleotide probe II was designed as follows: 30 pmol of the additional single-stranded oligonucleotide was spotted on the nucleic acid lateral flow. The strips were prepared.

[0148] 0.016 mg / ml -1 60 μl of the appropriate buffer containing carbon adsorbed to biotin-binding protein A buffer solution containing 0.5 pmol of a first oligonucleotide probe. Reactions were conducted to test the performance of lobes I and II. Reactions for II were constructed similarly, but 0.5 pmol of the second oligonucleotide probe II was added. A single stranded DNA target (representing at least one species in the amplified products obtained from the PCR reagent) was subjected to a variety of PCR reactions. (+++=1 pmol, ++=0.1 pmol, NTC=no target control) were added at 100 μl. After incubating at 45°C for 2 minutes, the entire reaction mix was transferred to the appropriate nucleic acid lateral freeze plate. Loaded on the lip.

[0149] Figure 10A shows a photograph of the lateral flow strip obtained in this experiment. The left panel shows the second The right panel shows the results using oligonucleotide probe I, and the right panel shows the results using a second oligonucleotide probe. Results are shown for the Tidprobe II. A black spot was visualized in the presence of the target. Stronger signals were observed for Reotide Probe II at all target levels.

[0150] (Example 6.2) Next, to demonstrate the versatility of the above embodiment of this method and its broad applicability, To achieve this, separate assays were designed for completely different target nucleic acids. The probe is probed with the relevant target nucleic acid, single-stranded DNA, in a manner similar to that described in Example 6.1. Again, two forms of the second oligonucleotide probe were designed: "I" and "II" and used various target levels (+++=1 pmol, ++=0.1 pmol, +=0.001 pmol). As shown in the photograph of the produced lateral flow strips shown in Figure 10B, More dramatic effects were observed in the two lowest target levels tested. Oligonucleotide probe I did not produce any signal, whereas the corresponding repeat oligonucleotide Nucleotide probe II showed clear positive results indicated by black spots of deposited carbon. produced sexual signals.

[0151] In this example, a second oligonucleotide detection portion containing repeated copies of a DNA sequence motif A significant improvement over lateral flow hybridization-based detection using This represents a 100-fold improvement in the sensitivity of nucleic acid lateral flow-based detection of the detector species. This demonstrates that the signal intensity is enhanced, the signal develops more rapidly, and This makes it easier to apply the present invention to applications involving rapid detection, for example, by nucleic acid lateral flow. The potential of the above embodiment of the invention is demonstrated. This illustrates the potential use of single-stranded oligonucleotides as detection moieties conjugated to

[0152] Example 7 (Use of RNA virus detection methods in clinical specimens) In this embodiment, the first oligonucleotide probe is added to the sample simultaneously with the amplification step a). The moiety that allows the second oligonucleotide probe to bind to the solid material is tribasic. a single stranded oligonucleotide containing four repeated copies of a base DNA sequence motif, An embodiment of the method in which the reverse complement of the single stranded oligonucleotide sequence is bound to a solid material. This study demonstrates the feasibility of using the method to detect RNA viruses in clinical specimens. In this study, we routinely used very low copy number RNA targets such as viral genome extracts. For example, using quantified viral genome extracts, the present inventors have Using the method of the invention, viruses with less than 100 genome-equivalent copies can be screened with a total time to result of less than 10 minutes. The detection is performed in 10 min and the amplification step a) takes less than 5 min. This remarkable speed and sensitivity makes it a valuable tool for diagnostic analysis. Thus, in this example, the inventors demonstrate the potential of the method for application in the field of disease. We developed an assay to detect the virulent single-stranded RNA virus and used it to detect the virulent single-stranded RNA virus in clinical specimens infected with the virus. The performance of the assay was demonstrated using

[0153] In the 5' to 3' direction: a synthetic stabilizing region of eight bases containing phosphorothioate bonds between each base ; 5-nt non-nicking restriction enzyme recognition sites; and in single-stranded RNA virus genomes A first hybridization sequence in the target nucleic acid designed to target a region A total length of 25 nucleotide bases including a 12-base hybridization region containing the reverse complementary sequence A first oligonucleotide primer was designed containing the same stabilizing region but with phosphorothioate. It does not contain a thioate bond and contains the same restriction enzyme recognition sequence, but is The hybridization region of 12 bases is capable of hybridizing with the reverse complementary strand of the target sequence. In this example, a second oligonucleotide primer was designed containing the first restriction enzyme The first and second restriction enzymes in the target nucleic acid are the same. The hybridization sequences are separated by 0 bases.

[0154] The oligonucleotide primers are designed using the target nucleic acid, thereby The nucleotide base downstream of the cleavage site on the reverse complementary strand becomes adenosine, resulting in an α-thiol dATP is utilized as the modified dNTP for use in this method. The modification is inserted by a strand-displacing DNA polymerase or reverse transcriptase to prevent cleavage of the reverse complementary strand. Stop.

[0155] In the 5' to 3' direction: the colorimetric dye of the first oligonucleotide probe, carbon nanotube a 5' biotin modification added during synthesis to allow binding to particles; an 8-base stabilization region; The restriction enzyme cleavage site in the first oligonucleotide probe is a nucleotide sequence added during synthesis. Restriction protected by phosphorothioate internucleotide bonds, but not by nicking enzymes A 5-base recognition sequence for the enzyme; hybridizes with at least one species in the amplification product. and 3' phosphate modification that prevents extension by strand-displacing DNA polymerases. A first oligonucleotide probe having a total length of 24 bases was designed, comprising:

[0156] In the 5' to 3' direction: downstream of the at least one species of first single-stranded detection sequence in the amplification product A 14 base region capable of hybridizing with a second single-stranded detection sequence; 5× thymidine bases a spacer comprising a reverse complement to which is immobilized on a lateral flow strip; A second oligonucleotide probe of 31 bases in length containing 4x repeats of a 3-base DNA sequence motif. A middle spacer containing 11× thymidine bases; a second oligonucleotide probe 12× repeats of a three-base sequence motif that is complementary to a three-base sequence motif of the gene We designed immobilized lateral flow printed oligonucleotides that are 47 bases long. In the 5' to 3' direction: a 5x triplet different from that on the second oligonucleotide probe. repeat sequence; an intermediate spacer containing 5× thymidine bases; a 3′ biotin base added during synthesis A lateral flow control oligonucleotide of 20 bases in length containing the molecule was designed. The oligonucleotide binds to its reverse complement on the lateral flow strip and is transported to the carbon lattice. Verify whether the RAL flow procedure is successful.

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

[0158] Figure 11 shows a photograph of a lateral flow strip obtained in the practice of this example. The reverse complement of the triplet repeat sequence of the second oligonucleotide probe is printed. Therefore, the position where a positive signal appears (+) and the position where the lateral flow was successfully performed are shown. A control oligonucleotide that will appear either positive or negative in the assay to verify whether the assay was successful. The positions of the reverse complement of the cytosine transfectant (CTL) are shown. The upper panel (+ve) shows the virus-positive clinical In the lower panel (-ve), we show the results obtained using a virus-negative sample. A distinct black line was present in each of the positive samples indicating the presence of the target nucleic acid, thereby Rapid detection of clinical specimens by the method of the present invention was demonstrated. No false positives were observed, e.g., 1 Complete absence of non-specific production of detector species through synthesis from or interprimer binding No false negatives were observed, demonstrating the robustness of the method and its presence in different clinical samples. Its sensitivity across different target nucleic acid copy number levels was demonstrated.

[0159] Example 8 (Performing the method at different temperatures) The method of the present invention is carried out efficiently over a wide range of temperatures and does not require cyclic changes in temperature. , any hot or warm start, preheat, or controlled temperature cool This example illustrates the performance of a typical assay over a range of different temperatures. Select an enzyme with the desired optimum temperature and perform melting of the hybridization after incorporation. Using temperature-reducing phosphorothioate bases, amplification can be performed over an unexpected wide range of temperatures. Assays that can be performed in a variety of ways and usually cover a low temperature range have been readily developed. In a separate experiment, an assay developed using the method of the present invention was performed in which the sample was wherein the temperature is maintained at 100° C. for 24 hours during amplification in step a) without the need to preheat the It is demonstrated that when the concentration is increased to 0.5, no decrease in performance is observed.

[0160] (Example 8.1) The first oligonucleotide probe is elongated by the DNA polymerase is blocked at the 3' end and cannot be cleaved by the first or second restriction enzyme, The assay was designed to utilize an embodiment of the method in which step a) is performed while simultaneously contacting the sample. In the 5' to 3' direction: a 7-base intermediate region; a recognition site for a restriction enzyme; and a target nucleic acid, a DNA target. The first hybridization sequence comprises an 11-base region capable of hybridizing to the first hybridization sequence. The first primer was designed to include the following: 5' to 3' direction: 7 bases in the middle region; the same restriction enzyme recognition site as the first restriction enzyme recognition site; and the reverse of the second hybridization sequence in the target nucleic acid. The second primer is modified to include a 12-base region capable of hybridizing with the complementary strand. Designed it.

[0161] In the 5' to 3' direction: 5' biotin modification; 6-base intermediate region; restriction containing a mismatch at position 2 The base of the recognition site of the enzyme; the first hybridization site in the target containing a G-clamp modification at position 6 a 10-base region capable of hybridizing with the 3′ phosphate modification (biotin); The first oligonucleotide probe was modified with a colorimetric dye, carbon nanoparticles, and The phosphate modification prevents its extension by strand-displacing DNA polymerases. A first oligonucleotide probe having a total length of 21 bases was designed to contain the sequence (

[0162] In the 5' to 3' direction: Hybridizes with the reverse complement of the second hybridization sequence in the target. 11 base region that can be dimerized; solid material of the second oligonucleotide probe The 4× thymidine base spacer and the 3-base sequence motif serve as binding enabling moieties. A second oligonucleotide probe was designed to contain 12 bases including the × repeat sequence. In the ' to 3' direction: 11 x thymidine base spacer; solid material of the second oligonucleotide and The 11x repeat sequence of the reverse complement to the three-base sequence motif that forms the moiety that allows binding of An additional single-stranded oligonucleotide was designed to contain a 33-base region containing the sequence. For oligonucleotide probes, 30 pmol of the additional single-stranded oligonucleotide A nucleic acid lateral flow strip was prepared by spotting the following:

[0163] 1.5 pmol of first primer; 1.0 pmol of second primer; 1 pmol of first oligonucleotide Otide probe: 60 μM Sp-dATP-α-S from Enzo Life Sciences; 60 μM dTTP each; dCTP, and dGTP; and various levels of target DNA (++=1 amol, +=10 zmol, NTC=non-target The reaction mixture was prepared in an appropriate buffer containing the target temperature (I = 3 7°C; II = 45°C, III = 50°C, and IV = 55°C) for 2 min, followed by a final 5 U of restriction enzyme and 5 U of Bacillus strand-displacing DNA polymerase to a final reaction volume of 25 μl. The reaction was then incubated for 5 min (T1) or 8 min (T2) with the relevant target After incubation, each reaction was diluted with 1.5 pmol of the second oligo Contains nucleotide probes and carbon adsorbed to 8 μg of biotin-binding protein The nucleic acid was then transferred to 75 μl of buffer solution and applied to the sample pad of a nucleic acid lateral flow strip. did.

[0164] FIG. 12A shows the results of lateral flow strips from experiments at each target level, temperature, and time point. The photograph shows that the distinct black lines observed are associated with detector species produced in the presence of target. At all temperatures, the carbon deposits were measured within 8 minutes at both target levels. In the presence of target, a very strong signal appears, and the method has a wide temperature range for efficient amplification. No nonspecific amplification was observed in the NTC samples. Strong amplification was observed after just 5 minutes, suggesting that the optimal temperature for this assay may be between 40°C and 50°C. It was shown to be highly

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

[0166] In the 5' to 3' direction: 5' biotin modification; 6-base intermediate region; restriction containing a mismatch at position 4 The enzyme's base recognition site; the site that hybridizes with the first hybridization region in the target. and a 12-base region capable of binding to the first oligonucleotide; and a 3' phosphate modification (biotin modification) The leutide probe can be combined with colorimetric dyes and carbon nanoparticles. The first oligonucleotide, which is 23 bases long and contains the nucleotide sequence 1, prevents its extension by strand-displacing DNA polymerase. A oligonucleotide probe was designed.

[0167] In the 5' to 3' direction: the 3-base reverse complement of the second hybridization sequence in the target. A 13-base region capable of hybridizing and a first and second hybridization sequence 10 base gap between rows; allows for binding of a second oligonucleotide probe to the solid material 3× thymidine base spacer and 4× repeats of the 3-base motif that act as a part of the The second oligonucleotide probe was designed to contain 12 bases including the 5' to 3' ends of In the direction: 11× thymidine base spacer; binding of the second oligonucleotide to the solid material 3 containing 12x repeats of the reverse complement of the 3-base sequence motif that forms the enabling portion An additional single-stranded oligonucleotide was designed to contain a 6-base region. For the oligonucleotide probe, 30 pmol of the additional single-stranded oligonucleotide was spotted. A nucleic acid lateral flow strip was prepared.

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

[0169] The reaction mixture was then incubated at 45°C for 6 minutes. The lateral flow strip was then loaded with 3 pmol of a second oligonucleotide probe. Figure 12B shows the lateral profiles obtained in the experiments under each temperature incubation condition. The clear black lines observed are the diffusion lines produced in the presence of the target. In amplification step a), the temperature is increased from 15°C to 45°C. No significant differences were observed in the augmented reactions. Amplification rates were obtained and no non-specific amplification was observed in the NTC samples.

[0170] This Example 8 shows that a lower optimal temperature profile is obtained using the method of the present invention compared to known methods. The NIRS 10001 has a 32-bit 32-bit LSB file and is capable of highly sensitive detection over an exceptionally wide temperature range. It is demonstrated that the assay can be easily developed. Also, the method of the present invention comprises the steps of: This can be done without using preheating to increase the temperature. Such features make the method very attractive for use in low-cost diagnostic devices. High temperatures and tight heat control make disposable or equipment-free devices commercially impractical. This imposes complex physical constraints that increase the cost of goods for such devices to a point where By avoiding the requirement of known methods to preheat the sample immediately, the method of the present invention is more It can be implemented in a simplified sequence with fewer user steps, and thus This will improve the ease of use of diagnostic devices and reduce the overall time to results.

[0171] Example 9 (Implementation of a method in which a target nucleic acid is obtained from double-stranded DNA by strand invasion) In this embodiment, the single-stranded target nucleic acid is subjected to any specific action to separate DNA strands, e.g. Temperature denaturation, bump primers, or additional enzymes (e.g., helicases or recombinases) ) is detected without any requirement for the use of a single stranded site in double stranded DNA. Demonstration of use. The method of the present invention is used to demonstrate the user steps, components or use in carrying out the method. Both single-stranded RNA and double-stranded DNA targets can be analyzed without any additional physical requirements imposed on the instrument. The ease with which the β-amyloids can be detected gives this method a high degree of versatility for use in diagnostic applications. Give.

[0172] Example 9.1. Amplification of Protein-Coding Regions in the Double-Stranded DNA Genome of a Viral Target We developed a double-stranded genome sequence as a biomarker for the presence of the virus in clinical diagnosis. The first oligonucleotide probe may be a nucleic acid sequence or an mRNA transcript. The extension by the DNA polymerase is blocked at the 3' end by the first or second restriction enzyme. and the probe is contacted with the sample simultaneously with carrying out step a). The assay was designed using the following oligonucleotide primers and oligonucleotides: The probes were designed to target the target molecule according to an approach similar to that described in the other examples. This was done without any gaps between the first and second hybridization sequences in the nucleic acid.

[0173] 4 pmol of the first oligonucleotide primer; 2 pmol of the second oligonucleotide primer 2 pmol of first oligonucleotide probe; 60 μM from Enzo Life Sciences Sp-dATP-α-S; 60 μM each of dTTP, dCTP, and dGTP; 60 μg of biotin-binding protein and double-stranded DNA target (I) or single-stranded RNA target (II) or no target. The reaction mixture was prepared in an appropriate buffer containing 100% ethanol. The reaction mixture was incubated at 45°C for 2 minutes. After incubation, the final 20 U of restriction enzyme, 20 U of Bacillus strand-displacing DNA polymerase, and 2 The reaction was started by adding 5 U of reverse transcriptase to a final reaction volume of 100 μl. Addition of enzyme The reaction mixture was then incubated at 45° C. for 7 minutes.

[0174] After incubation, 1.5 pmol of a second oligonucleotide probe was added to each reaction. The entire reaction volume was transferred to the sample pad of a nucleic acid lateral flow strip. A flow control target was also added to all samples. Figure 13A shows the results obtained in this experiment for each target. A photograph of a lateral flow strip is shown. The distinct black lines observed are those that occur in the presence of target. The fainter signals correspond to carbon deposits that bind to the detector species produced. , corresponds to lower target levels (+) than higher target levels (++). No differences in amplification rates were observed among the A targets.

[0175] Example 9.2: The assay was performed to identify single stranded DNA in the c.2.5 megabase double stranded DNA genome of a bacterial pathogen. The first oligonucleotide probe is designed for a target nucleic acid. The extension by the first or second restriction enzyme is blocked at the 3' end, and the first or second restriction enzyme cannot cleave the protein. The assay can be carried out using an embodiment of the method in which the probe is contacted with the sample simultaneously with carrying out step a). The oligonucleotide primers and probes were designed according to the methods described elsewhere. According to an approach similar to that described in the previous embodiment, the first and second Hybridization was performed with a gap of 4 bases between the hybridization sequences.

[0176] 2 pmol of the first oligonucleotide primer and 0.5 pmol of the second oligonucleotide Reactions were prepared in an appropriate buffer containing the primers. In fact, two single-stranded target nucleic acids are added simultaneously, and a second reciprocal process occurs at the same time. Here, the second oligonucleotide primer for detecting the target nucleic acid is A first oligonucleotide primer for detecting a second target nucleic acid, This fact has little effect on the performance of the method. 60 μM Sp-dATP-α-S from Enzo Life Sciences; 0 μM dTTP, dCTP, and dGTP; 15 μg of biotin-binding protein adsorbed to carbon as well as cells containing the target at various levels (++=1 amol; +=10 zmol; NTC=no target control). In addition, a specificity control reaction containing 1 amol of E. coli genomic extract was also performed. The assembled reaction mixture was incubated at 45°C for 3 minutes, and then a final 4 U of restriction enzyme, and 2 U of Bacillus strand-displacing DNA polymerase to a final reaction volume of 25 μl. After addition of the enzyme, the reaction was incubated at 45° C. for 6 minutes.

[0177] After incubation, 75 μl of buffer containing 3 pmol of the second oligonucleotide probe was added. Buffer solution was added to each reaction, and the entire volume was then applied to the sample pad of the nucleic acid lateral flow strip. Figure 13B shows the photographs of the lateral flow strips obtained in this experiment for each target. The bright spots correspond to the deposition of carbon bound to the detector species produced in the presence of the target. A clear black line was observed at both target levels tested. In the presence of E. coli genomic DNA, no nonspecific signals were observed, and the method was demonstrated to be effective in only 6 The new technology allows for specific detection of complex double-stranded DNA genomes at clinically relevant copy numbers within minutes. It has been proven that this is the case.

[0178] In this example, the method of the present invention can be readily used to detect single-stranded nucleic acid targets in double-stranded DNA. Remarkably, we demonstrate that the same target sequence in a single-stranded RNA target and in double-stranded DNA can be expressed by For detection of sequences, no specific action to separate the DNA double strands, such as temperature denaturation, is required. Similar amplification rates were observed without any restriction. Instead, the single-stranded site was located at the first oligonucleotide. sufficiently exposed for hybridization and extension of the primer, leading to "strand invasion" The method begins with a transient opening of one or more DNA base pairs in the double-stranded DNA. Hybridization and extension of the 3' hydroxyl of the first oligonucleotide primer This occurs sufficiently to allow for thermal denaturation in assays of double-stranded nucleic acids. This is in contrast to known methods such as SDA, which utilizes single-stranded DNA and bump primers. The method of the present invention allows easy detection of targets in double-stranded DNA as well as targets in single-stranded RNA. The ability to use the method is of great value in its diagnostic applications, e.g., in the detection of bacterial, fungal, and and confers a high degree of versatility for use in the detection of viral pathogens. Additional user steps, enzymes, components, or substances that complicate the detection of organisms with double-stranded DNA genomes The fact that no physical constraints are required makes this method feasible for use with simplified, low-cost diagnostic equipment. For example, the amplification method reported in the known method is particularly suitable for the detection of The requirement for heat denaturation prior to implementation of the method requires costly additional components and the commercial value of such devices is questionable. This increases the cost of products and the overall time to results. This means that a device that requires no equipment is not practical.

[0179] Example 10 (Comparison of the method of the present invention with known methods) In this example, detection of viral targets was performed using a known method disclosed in WO2014 / 164479. A comparative evaluation of the method of the invention against known methods is presented. Known methods require a nicking enzyme and one or more The method of the present invention is fundamentally different from the method of the present invention in that it does not require the use of the above modified dNTPs. The method is demonstrated to have excellent sensitivity and specificity.

[0180] For this comparative evaluation, the method of the present invention was used to identify viral targets having single-stranded RNA genomes. The first oligonucleotide probe was used to detect the DNA fragment. The extension by the restriction enzyme is blocked at the 3' end, and the cleavage is prevented by the first or second restriction enzyme. By using an embodiment of the method in which the probe is contacted with the sample simultaneously with carrying out step a), The assay was designed. Oligonucleotide primers and oligonucleotide probes The probe was designed by following an approach similar to that described in other examples, The hybridization was performed with a gap of 6 bases between the first and second hybridization sequences in .

[0181] For assays using known methods, the equivalent primers used in the method of the present invention are Contains the same 6-base intermediate region at the 5' end and the same hybridization region at the 3' end Similar primers were designed. Thus, to allow for a rigorous comparison of the methods, Consistency between assays was ensured as much as possible. However, the bases of the restriction enzyme recognition sites were , WO2014 / 164479, Nt.BbvCI (See Example 5 on pages 20-21.)

[0182] (Example 10.1) In the first example, reactions for each method were carried out using equal primer ratios. For the method of the present invention: 2 pmol of the first oligonucleotide primer; 2 pmol of the second Oligonucleotide primers; 1.6 pmol of the first oligonucleotide probe; Enzo Li 60 μM Sp-dATP-α-S from Fe Sciences; 60 μM each of dTTP, dCTP, and dGTP; and Various levels (+++=10 zmol; ++=100 copies; +=10 copies; NTC=no target control) of Reactions were prepared in appropriate buffers containing virus genomic RNA extract as the target. The reaction mixture was preincubated at ambient temperature (20°C) for 5 min, and then 5 U of restriction enzyme, 5 U of of Bacillus strand-displacing DNA polymerase, and 10 U of reverse transcriptase in a final reaction volume of 25 μl. After addition of the enzyme, the reaction was incubated at 45°C for 8 min (T1) or 15 min (T2). (T2) incubation. After incubation, each reaction solution was added with 60 μg of Add 100 μl of carbon adsorbed with biotin-binding protein to the sample pack. A nucleic acid lateral flow strip containing 1.5 pmol of a second oligonucleotide probe on the It was moved to the pool.

[0183] For known methods: 6.25 pmol of first oligonucleotide primer; 6.25 pmol of a second oligonucleotide primer; 200 μM each of dATP, dTTP, dCTP, and dGTP; and at various levels (+++=10 zmol; ++=100 copies; +=10 copies; NTC=no target control). Reactions were prepared in appropriate buffers containing viral genomic RNA extracts as targets. The reaction mixture was preincubated at ambient temperature (20°C) for 5 min, then 4 U of Nt.BbvCI, 20 10 U of Bst large fragment DNA polymerase, and 10 U of M-MuLV reverse transcriptase in a final reaction volume of 25 μl. After addition of the enzyme, the reaction was incubated at 45°C for 8 min (T1) and 15 min (T2). (T2) incubation. After incubation, 60 μg of biotin-bound protein was added. 75 μl buffer containing adsorbed carbon and 5 pmol of the first oligonucleotide probe Add 100 μl of 100 μl of 100 μl of 100 μl of 2nd oligonucleotide to each reaction and deposit 5 pmol of 2nd oligonucleotide on the sample pad. The nucleic acid was then transferred to a lateral flow strip containing the probe.

[0184] In FIG. 14A, experiments using the method of the invention (I) at various target levels and at the indicated time points. and a photograph of a lateral flow strip obtained in an experiment using the known method (II). The black lines observed are the carbohydrates bound to the detector species produced in the presence of the target. It is possible to observe any signal at all using known methods. It took several attempts to achieve this, and the enzymes and buffers, as well as significantly higher levels of It was necessary to use a specific combination of primers, dNTPs, and enzymes. Using (I), even at the shortest time point after only 8 minutes without preheating, only 10 copies were Unambiguous identification of detector species produced, even at the lowest target levels Even after efforts to optimize known methods that are not obvious to those skilled in the art, Only a faint signal was observed at the highest target level (+++ = 10 zmol) and the longest time point (15 min). It wasn't noticed.

[0185] (Example 10.2) Furthermore, after extensive and non-obvious trials, the results shown in Example 10.2 were A 2:1 ratio of the first and second oligonucleotides was used with a very high concentration of the first primer. It is possible to improve the performance of known methods, but only by using primers. The method of the present invention was carried out as described in Example 10.1. For the method, reactions were run with the first oligonucleotide primer level increased to 12.5 pmol. The experiments were carried out as described in Example 10.1, except that the following target levels were obtained in each case: : +++=1 zmol; ++=100 copies; +=10 copies; NTC=no target control was used.

[0186] In FIG. 14B, experiments using the method of the invention (I) at various target levels and at the indicated time points. and a photograph of a lateral flow strip obtained in an experiment using the known method (II). The black lines observed are the carbohydrates bound to the detector species produced in the presence of the target. Again, the method (I) of the present invention corresponds to the deposition of ions at only 1 with visible signal even at very low target levels of 0 copies of target It has been demonstrated that remarkable speed can be achieved using known methods. (+++=1 zmol) only a faint signal was observed at the longest time point (15 min). A very faint signal was also visible in the 100 copy sample. The very high oligonucleotide primers required for the method to work in TC strips Faint signals that may correspond to non-specific products as a result of the immersion and enzyme levels. These data are consistent with those in WO2014 / 164479, where the incubation time was 30 minutes. This is consistent with the data reported in the previous paper. The requirement for extraordinarily high primer levels for amplification of two or more amplicons in the same sample This severely limits its potential applicability for the detection of different targets. There is considerable scope for further increasing the total primer level without exacerbating the problems associated with Because it is always limited.

[0187] This Example 14 demonstrates that amplification is much faster, more sensitive, and produces clearer results. The present invention goes beyond the known method disclosed in WO2014 / 164479 in that a signal of This demonstrates the remarkable superiority of the method proposed here. The method described uses only 100 copies of target, with the highest target level using 60-fold target levels. The results showed that the 15 minute reaction produced a stronger signal than can be produced in known methods, which can be performed in a 15 minute incubation at 37°C. The advantage of the method of the present invention over this known method is that it does not use a restriction enzyme that is a nicking enzyme. that it requires a different class of enzymes, which enhances the sensitivity and specificity of the amplification. This results from the requirement of using one or more modified dNTPs, such as phosphorothioate bases. Furthermore, one of the first and second oligonucleotide probes is The extension by the restriction enzyme is blocked at the 3' end, and the nucleic acid is cleaved by the first or second restriction enzyme. and contacting the sample simultaneously with carrying out step a), effectively coupling amplification with signal detection. and allows efficient sequence-based hybridization during the formation of the detector species. These advantages facilitate the increased specificity resulting from the fusion of the The method of the present invention has applications in the field of diagnostics and is a simplified, ultra-fast, user-focused, low Ideal for the development of cost-effective diagnostic devices, such as disposable or instrument-free molecular diagnostic testing devices Throughout the following specification and claims, unless the context otherwise requires: The terms "comprise", "comprises", "comprising" and "comprising" " implies including the stated integer, step, group of integers, or group of steps, but It will be understood that this does not exclude any other integer, step, group of integers, or group of steps. cormorant.

[0188] Further aspects of the present invention include the following: Embodiment 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is blocked by the presence of one or more modifications incorporated into the reverse complementary strand by a DNA polymerase the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by DNA polymerase the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) subjecting the amplification product of step a) to: i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is linked to a moiety that enables binding; and hybridizing said first and second probes with said at least one species in said amplification product. the contacting, wherein the detector species is produced by photoredization; and c) detecting the presence of the detector species produced in step b), the presence of a vector species indicates the presence of the target nucleic acid in the sample. , the method. Aspect 2. One of the first and second oligonucleotide probes is a DNA polymerase The 3'-terminus of the nucleic acid is blocked from being extended by the first or second restriction enzyme, and the nucleic acid cannot be cleaved by the first or second restriction enzyme. The method described in Example 1. Aspect 3. The one oligonucleotide probe has one or more sequence mismatches and / or one or more The first or second restriction enzyme is unable to amplify the nucleic acid because of the presence of the above modifications, e.g., phosphorothioate bonds. The method of embodiment 2, wherein the nucleic acid sequence is rendered incapable of being cleaved by the nucleic acid sequence. Aspect 4. The one oligonucleotide probe is contacted with the sample simultaneously with carrying out step a). The method according to embodiment 2 or 3, In the step a), the sample further includes: (A) a 5' to 3' region of the first restriction enzyme recognition site; The first hybridization sequence and cleavage site in the target nucleic acid are hybridized with the first hybridization sequence in the target nucleic acid. It contains one strand of the region that can hybridize to the 3' end and is extended by DNA polymerase. and / or (B) a third oligonucleotide primer, The recognition sequence and cleavage site of the second restriction enzyme and the second hybridization site in the target sequence. a region capable of hybridizing to the reverse complement of a target sequence, A fourth oligonucleotide primer, the 3' end of which is blocked from extension by NA polymerase. The method according to any one of embodiments 1 to 4, wherein the contacting is with Aspect 6. When present, the third oligonucleotide primer is a primer that is complementary to the first oligonucleotide primer. provided in excess of the oligonucleotide primer and, if present, 6. The method of embodiment 5, wherein the first primer is provided in excess of said second oligonucleotide. 7. The method according to any one of claims 1 to 6, wherein the one or more modified dNTPs are α-thiol modified dNTPs. How to. Aspect 8. The method according to any one of aspects 1 to 7, wherein the first and second restriction enzymes are the same restriction enzyme. method. Embodiment 9. The method according to any one of embodiments 1 to 8, wherein two or more of steps a), b), and c) are carried out simultaneously. How to. Embodiment 10. The method of any one of embodiments 1 to 9, wherein step (a) is carried out at a temperature of 50° C. or less. 11. The moiety that enables detection of the first oligonucleotide probe is a colorimetric dye or a fluorometric dye, or a moiety capable of binding to a colorimetric or fluorometric dye; The method according to any one of aspects 1 to 10, wherein the active ingredient is biotin. 12. Any of the above embodiments 1 to 11, wherein the detector species is detected by a change in an electrical signal. Please select one of the methods. Aspect 13. The detectable moiety of the first oligonucleotide probe is attached to a substrate. The enzyme is an enzyme that produces a detectable signal, such as a colorimetric or fluorometric signal, after contact with the The method according to any one of aspects 1 to 12, Embodiment 14. The moiety of the second oligonucleotide probe that enables binding to a solid material comprises: The method according to any one of aspects 1 to 13, wherein the oligonucleotide is a single-stranded oligonucleotide. Aspect 15. The sequence of the single-stranded oligonucleotide portion is a sequence of three or more 2-4 base DNA sequence motifs. The method of embodiment 14, comprising repeated copies. In embodiment 16, the presence of the detector species in step c) is detected by nucleic acid lateral flow. The method according to any one of aspects 1 to 15, Aspect 17. The nucleic acid lateral flow is a solid material of the second oligonucleotide probe. One or more nucleic acids capable of sequence-specific hybridization with a moiety that allows for the binding of The method of embodiment 16, wherein an acid is utilized. Embodiment 18. Step c) of colorimetric or electrochemical determination using carbon or gold, preferably carbon The method of any one of embodiments 1 to 17, wherein a chemical signal is generated. 19. The first and / or second oligonucleotide primers comprise a restriction enzyme recognition sequence and a cleavage site. 19. Any one of embodiments 1 to 18, further comprising a stabilizing sequence, for example, 5 bases long, at the 5' end upstream of the site. How to. Aspect 20. The hybridizing region of the first and / or second oligonucleotide primer is 20. The method according to any one of aspects 1 to 19, wherein the nucleic acid sequence is 9 to 16 bases in length. Aspect 21. One of the first and second oligonucleotide primers is more selective than the other primer. The method according to any one of embodiments 1 to 20, wherein the antibody is provided in excess of the antibody. Aspect 22: The first and second hybridization sequences in the target nucleic acid are composed of 0 to 6 bases. The method according to any one of embodiments 1 to 21, wherein the first and second electrodes are spaced apart. Aspect 23: The first and second hybridization sequences in the target nucleic acid are composed of 3 to 6 bases. The method according to any one of embodiments 1 to 21, wherein the first and second electrodes are spaced apart. In the step b), the first or second one of the at least one species in the amplification product is Any one of aspects 1 to 23, wherein the strand detection sequence comprises a sequence corresponding to 3 to 6 bases defined in aspect 23. The method described herein is as follows. 25. The method of any one of claims 1 to 24, wherein the level of the target nucleic acid in the sample is quantified in step c). or one of the methods described. 26. The target nucleic acid comprises single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA. Single-stranded DNA, including single-stranded RNA or single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA The method according to any one of embodiments 1 to 25, 27. The single-stranded DNA is cleaved by a nuclease, such as a restriction endonuclease or an exonuclease. It can be obtained from double-stranded DNA by using nuclease III or from single stranded DNA by using reverse transcriptase. 30. The method of embodiment 26, wherein the stranded RNA is obtained from a single stranded RNA. 28. The presence of two or more different target nucleic acids of defined sequence is detected in the same sample. The method described in any one of 1 to 27. 29. The sample is a biological sample, e.g., a nasal or nasopharyngeal swab or aspirate, blood, or The method according to any one of aspects 1 to 28, wherein the sample is a blood-derived sample, or urine. 30. The target nucleic acid is viral or derived from viral nucleic acid material, and is not bacterial or Circulating cell-free DNA derived from bacterial nucleic acid material and released from cancer cells or fetal cells; 30. The method according to any one of aspects 1 to 29, which is a microRNA or is derived from a microRNA. Law. 31. The method of any one of claims 1 to 30, wherein the target nucleic acid comprises an epigenetic modification site, such as methylation. The method described herein is as follows. 32. Detection of the target nucleic acid is used for diagnosis, prognosis, or monitoring of a disease or disease state. The method according to any one of embodiments 1 to 31, 33. The disease is, but is not limited to, HIV, influenza, RSV, rhinovirus, Virus, Norovirus, Tuberculosis, HPV, Meningitis, Hepatitis, MRSA, Ebola, Clostridium difficile, Epstein-Barr virus, malaria, plague, polio, chlamydia, Infectious diseases, including herpes, gonorrhea, measles, mumps, rubella, cholera, or smallpox 33. The method of embodiment 32. 34. The disease is, but is not limited to, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, or the like. Cancer including adenocarcinoma, liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, or melanoma. 33. The method of embodiment 32. Aspect 35. The detection of the target nucleic acid is performed in human genetic testing, prenatal testing, blood contamination screening, etc. 35. The method according to any one of embodiments 1 to 34, for use in drug discovery, pharmacogenomics, or pharmacokinetics. method. 36. The sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biological sample. The method according to any one of embodiments 1 to 35, which is a protective sample. Embodiment 37. A kit comprising: a) a first oligonucleotide primer and a second oligonucleotide primer; Thus, the first primer has a restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction, and A single strand of a defined sequence hybridizes to a first hybridization sequence in a target nucleic acid. and the second primer comprises a region capable of cleaving a restriction enzyme recognition region in the 5' to 3' direction. A sequence and cleavage site and a second sequence upstream of the first hybridization sequence in the target nucleic acid. A region capable of hybridizing with the reverse complement of the hybridization sequence of , the first oligonucleotide primer and a second oligonucleotide primer; b) a nicking enzyme that recognizes the recognition sequence of the first primer and forms the cleavage site The second primer is not a first restriction enzyme and a nicking enzyme that can cut the a second restriction enzyme capable of recognizing said recognition sequence and cleaving said cleavage site; c) strand-displacing DNA polymerase; d) dNTPs, e) one or more modified dNTPs; f) a hybridization region of one of the first and second oligonucleotide primers; A first oligonucleotide having a degree of complementarity to the first oligonucleotide and attached to a moiety that allows its detection. tide probe; and g) the reverse phase of the hybridizing region of the other of the first and second oligonucleotide primers a portion that has a degree of complementarity with the complementary strand and allows for binding to the solid material or to the solid material; a second oligonucleotide probe attached thereto. 38. The kit according to embodiment 37, further comprising a means for detecting the presence of the detector species. . Aspect 39. The first oligonucleotide primer and / or the second oligonucleotide primer a primer, and / or the first restriction enzyme, and / or the second restriction enzyme, and / or said DNA polymerase, and / or said dNTPs, and / or said one or more modified dNTPs, and / or The first oligonucleotide probe and / or the second oligonucleotide probe The method according to any one of the aspects 2, 3, 7, 8, 11, 13 to 17, 19, 20, or 22 to 24, The kit according to embodiment 37 or 38, Embodiment 40. A third and / or fourth oligonucleotide primer as defined in embodiment 5 or 6. 40. The kit according to any one of embodiments 37 to 39, further comprising: 41. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: : a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; and inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) Purifying the amplification product of step a): i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is linked to a moiety that enables binding; and one of the first and second oligonucleotide probes is a DNA polymerase The extension by ferrocene is blocked at the 3' end, and the 3' end is cleaved by the first or second restriction enzyme. and the first and second probes cannot be hybridized with the at least one species in the amplification product. said contacting, wherein hybridization produces a detector species; and To c) detecting the presence of the detector species produced in step b), wherein the presence of a target species indicates the presence of the target nucleic acid in the sample; wherein said first or second oligonucleotide probe as defined in step b) is subjected to step a) The method wherein said sample is contacted with said

[0189] All patents and patent applications referred to herein are incorporated by reference in their entirety. It is being read.

Claims

1. 1. A method for detecting the presence of a single-stranded target nucleic acid of defined sequence in a sample, comprising: a) separating the sample by: i. a first oligonucleotide primer and a second oligonucleotide primer The first primer comprises, in the 5' to 3' direction, a restriction enzyme recognition sequence and a cleavage site, and A region capable of hybridizing to a first hybridization sequence in the target nucleic acid. and the second primer contains in the 5' to 3' direction a restriction enzyme recognition sequence and a cleavage site. The cleavage site and a second hybridization sequence upstream of the first hybridization sequence in the target nucleic acid. It contains one strand of a region capable of hybridizing with the reverse complement of the transcription sequence. , the first oligonucleotide primer and a second oligonucleotide primer; ii. strand-displacing DNA polymerase; iii. dNTPs; iv. one or more modified dNTPs; v. A nicking enzyme that is not a nicking enzyme but recognizes the recognition sequence of the first primer and and cleaves only the first strand of the primer at the cleavage site if the cleavage site is double stranded. a first restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; is inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the first restriction enzyme; and vi. a nicking enzyme that recognizes the recognition sequence of the second primer and sequence and cleaves only the second strand of the primer at the cleavage site if the cleavage site is double stranded a second restriction enzyme capable of cleaving the reverse complementary strand using the one or more modified dNTPs; and inhibited by the presence of one or more modifications incorporated into the reverse complementary strand by the DNA polymerase. the second restriction enzyme; and producing an amplification product in the presence of the target nucleic acid without the use of temperature cycling. To do so; b) PCR using the amplification product of step a): i. hybridizes to a first single-stranded detection sequence of at least one species in the amplification product; A first oligonucleotide probe capable of detecting the target gene and having a moiety that allows its detection. Robe; and ii. a first single-stranded detection sequence upstream or downstream of the at least one species in the amplification product; 2, and can hybridize to a solid material or a bond to the solid material. a second oligonucleotide probe, which is linked to a moiety that enables binding; contacting said first and second amplicons with said at least one species in said amplification product; Hybridization of the lobes produces a detector species. and c) detecting the presence of the detector species produced in step b), the presence of a target species indicates the presence of the target nucleic acid in the sample. The method comprising:

2. One of the first and second oligonucleotide probes binds to the DNA polymerase. The 3' end of the nucleic acid is blocked from being extended by the first or second restriction enzyme, and cannot be cleaved by the first or second restriction enzyme. The method described in item 1.

3. The blocked oligonucleotide probes may contain one or more sequence mismatches and / or one or more The first or second restriction enzyme is unable to amplify the nucleic acid because of the presence of the above modifications, e.g., phosphorothioate bonds. The method of claim 2, wherein the nucleic acid sequence is rendered incapable of being cleaved by the nucleic acid sequence.

4. The blocked oligonucleotide probe is contacted with the sample simultaneously with carrying out step a). The method according to claim 2 or 3,

5. The blocked oligonucleotide probe comprises an additional region, The 3' end of the species in the amplification product to which the nucleotide probe hybridizes is the strand displacement type The method according to any one of claims 2 to 4, which is capable of being extended by a DNA polymerase. 。

6. In step a), the sample further contains: (A) a recognition sequence of the first restriction enzyme in the 5' to 3' direction; a sequence and cleavage site and hybridizes to the first hybridization sequence in the target nucleic acid It contains one strand of a region that can be extended and is blocked at the 3' end by DNA polymerase. and / or (B) a third oligonucleotide primer, The recognition sequence and cleavage site of a second restriction enzyme and the second hybridization sequence in the target nucleic acid. The DNA fragment comprises one strand of a region capable of hybridizing with the reverse complement of the target sequence, A fourth oligonucleotide primer is joined to the 3′ end of the primer, which is blocked at the 3′ end from being elongated by the ribozyme. The method according to any one of claims 1 to 5, wherein the

7. If present, the third oligonucleotide primer is and if present, said fourth oligonucleotide primer is provided in excess of said first primer. The method of claim 5, wherein the primer is provided in excess of the second oligonucleotide.

8. The one or more modified dNTPs according to any one of claims 1 to 7 are α-thiol modified dNTPs. method.

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

10. The method according to any one of claims 1 to 9, wherein two or more of steps a), b) and c) are carried out simultaneously. method.

11. The process according to any one of claims 1 to 10, wherein step a) is carried out at a temperature below 50°C.

12. Starting ambient temperature, for example in the range of 15-30°C, increasing to a temperature in the range of 40-50°C, etc.

12. The method according to claim 1, wherein the temperature is increased during the implementation of step a).

13. The detectable moiety of the first oligonucleotide probe may be a colorimetric dye or or a fluorometric dye, or a moiety capable of binding to a colorimetric or fluorometric dye, e.g. The method according to any one of claims 1 to 12, wherein the ligand is biotin.

14. Any of claims 1 to 13, wherein the detector species is detected by a change in an electrical signal. or the method described in paragraph 1.

15. The detectable portion of the first oligonucleotide probe is contacted with a substrate. The enzyme produces a detectable signal, e.g., a colorimetric or fluorescent signal. The method of any one of 1 to 14.

16. The portion of the second oligonucleotide probe that enables binding to a solid material is The method of any one of claims 1 to 15, wherein the oligonucleotide is a stranded oligonucleotide.

17. The sequence of the single-stranded oligonucleotide portion is a repeat of three or more DNA sequence motifs of 2-4 bases.

17. The method of claim 16, further comprising:

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

19. The nucleic acid lateral flow is a method for binding the second oligonucleotide probe to a solid material. One or more nucleic acids capable of sequence-specific hybridization with a portion that allows the binding 20. The method of claim 18, wherein

20. Step c) is carried out by colorimetry or electrochemistry using carbon or gold, preferably carbon. The method of any one of claims 1 to 19, wherein a signal is generated.

21. The first and / or second oligonucleotide primers include a restriction enzyme recognition sequence and Any of claims 1 to 20, comprising a stabilizing sequence, for example 5 or 6 bases long, upstream of the cleavage site. or the method described in paragraph 1.

22. The hybridization region of the first and / or second oligonucleotide primer is 9 to 16 The method according to any one of claims 1 to 21, wherein the length is base length.

23. One of the first and second oligonucleotide primers is in excess of the other primer. The method according to any one of claims 1 to 22, wherein the

24. The first and second hybridization sequences in the target nucleic acid are separated by 0 to 15 bases. The method according to any one of claims 1 to 23,

25. The first and second hybridization sequences in the target nucleic acid are separated by 3 to 15 bases. The method according to any one of claims 1 to 24,

26. In step b), the first or second single-stranded detection of the at least one species in the amplification product The output sequence comprises a sequence of at least 3 bases corresponding to the 3 to 15 bases defined in claim 24. The method according to any one of items 1 to 25.

27. 27. The method according to claim 1, wherein the level of the target nucleic acid in the sample is quantified in step c). The method described.

28. The target nucleic acid includes single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double-stranded DNA. A, or single-stranded DNA, including single-stranded DNA derived from single-stranded RNA and single-stranded DNA derived from double-stranded DNA. The method of any one of claims 1 to 27.

29. 29. The method of claim 28, wherein the single-stranded target nucleic acid is a single-stranded DNA obtained from a double-stranded DNA by strand invasion. How to.

30. The single-stranded DNA may be subjected to a nuclease, such as a restriction endonuclease or an exonuclease. It can be derived from double-stranded DNA by using Reverse Transcriptase III or from single-stranded DNA by using Reverse Transcriptase.

30. The method of claim 28, wherein the method is obtained from RNA.

31. The presence of two or more different target nucleic acids of defined sequence is detected in the same sample.

30. The method of any one of claims 2 to 30.

32. The sample may be a biological sample, such as a nasal or nasopharyngeal swab or aspirate, blood or 32. The method of any one of claims 1 to 31, wherein the sample is a fluid-derived sample or urine.

33. The target nucleic acid is derived from a virus or viral nucleic acid material and is bacterial or It is circulating cell-free DNA derived from nucleic acid material and released from cancer cells or fetal cells, and is The method of any one of claims 1 to 32, wherein the RNA is a loRNA or is derived from a microRNA.

34. Detection of the target nucleic acid is for the diagnosis, prognosis, or monitoring of a disease or disease state. The method according to any one of claims 1 to 33, wherein said method comprises the steps of:

35. The disease may be, but is not limited to, HIV, influenza, RSV, rhinovirus, , Norovirus, Tuberculosis, HPV, Meningitis, Hepatitis, MRSA, Ebola, Clostridium diff Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes infectious diseases, including rabies, gonorrhea, measles, mumps, rubella, cholera, or smallpox; Item 35. The method according to item 34.

36. The disease includes, but is not limited to, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, The cancer is selected from the group consisting of liver cancer, bladder cancer, leukemia, esophageal cancer, ovarian cancer, renal cancer, gastric cancer, and melanoma. Item 35. The method according to item 34.

37. The detection of the target nucleic acid is used in human genetic testing, prenatal testing, blood contamination screening, drug The method according to any one of claims 1 to 36, for use in physicogenomics or pharmacokinetics. 。

38. The sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample, or a biodefense sample. The method of any one of claims 1 to 37, wherein the

39. 13. A kit comprising: a) a first oligonucleotide primer and a second oligonucleotide primer; Thus, the first primer has a restriction enzyme recognition sequence and a cleavage site in the 5' to 3' direction, and A single strand of a defined sequence hybridizes to a first hybridization sequence in a target nucleic acid. and the second primer comprises a region capable of cleaving a restriction enzyme recognition region in the 5' to 3' direction. A sequence and cleavage site and a second sequence upstream of the first hybridization sequence in the target nucleic acid. A region capable of hybridizing with the reverse complement of the hybridization sequence of , the first oligonucleotide primer and a second oligonucleotide primer; b) a nicking enzyme that recognizes the recognition sequence of the first primer and forms the cleavage site A first restriction enzyme capable of cleaving, and not a nicking enzyme, the second primer a second restriction enzyme capable of recognizing the recognition sequence and cleaving the cleavage site; c) strand-displacing DNA polymerase; d) dNTPs, e) one or more modified dNTPs; f) detecting at least one species of first single-stranded detection signal in the amplification product produced in the presence of the target nucleic acid; is linked to a moiety capable of hybridizing to the target sequence and allowing its detection; a first oligonucleotide probe; and g) a second single-stranded detection sequence upstream or downstream of the first single-stranded detection sequence of the at least one species in the amplification product; A single stranded detection sequence is hybridizable to the solid material or to the solid material. a second oligonucleotide probe coupled to an enabling moiety. 。

40. One of the first and second oligonucleotide probes binds to the DNA polymerase. at the 3' end, e.g., one or more sequence mismatches and / or one or more modifications, e.g. For example, a fragment that is cleaved by the first or second restriction enzyme due to the presence of a phosphorothioate bond.

40. The kit of claim 39, wherein the kit is incapable of

41. One of the first and second oligonucleotide probes is Complementarity of 5 or more bases with the hybridization region of the primer or the first or second primer 41. The kit of claim 39 or 40, comprising a reverse complement of the hybridizing region of said mer.

42. The first oligonucleotide probe is a first oligonucleotide probe having a first and a second oligonucleotide probe. The second oligonucleotide has a complementarity of 5 bases or more with one of the hybridizing regions of the first oligonucleotide. The oligonucleotide probe is a hybrid of the other of the first and second oligonucleotide primers. The kit of claim 41, wherein the nucleic acid sequence has 5 or more bases of complementarity with the reverse complement of the nucleic acid sequence of the nucleic acid region.

43. and means for detecting the presence of detector species produced in the presence of said target nucleic acid. The kit according to any one of claims 39 to 42, comprising:

44. The target nucleic acid in at least one species of the amplification product, the first oligonucleotide, and / or the second oligonucleotide primer, and / or the first and / or said second restriction enzyme, and / or said DNA polymerase, and / or the dNTP, and / or the one or more modified dNTPs, and / or the first oligonucleotide protease and / or said second oligonucleotide probe, and / or said first or second The single-stranded detection sequence of any one of claims 5, 8, 9, 13, 15 to 17, 21 to 26, or 28 to 33. The kit according to any one of claims 39 to 43, as defined above.

45. A third and / or fourth oligonucleotide primer as defined in claim 5 or 6. The kit of any one of claims 39 to 44, further comprising:

46. A device comprising the kit of any one of claims 39 to 45.

47. 47. The device of claim 46, which is a powered device.

48. 48. Apparatus according to claim 46 or 47, comprising heating means.

49. The device of any one of claims 46 to 48, which is a disposable diagnostic device.

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