WO2021210779A1

WO2021210779A1 - Method for detecting target nucleic acid using self-priming hairpin-utilized isothermal amplification

Info

Publication number: WO2021210779A1
Application number: PCT/KR2021/002664
Authority: WO
Inventors: 박현규; 송자연; 정유진; 이서영
Original assignee: 한국과학기술원; 재단법인 바이오나노헬스가드연구단
Priority date: 2020-04-14
Filing date: 2021-03-04
Publication date: 2021-10-21
Also published as: KR20210127375A; KR102359986B1

Abstract

The present invention relates to a method for detecting a target nucleic acid using a self-priming hairpin-utilized isothermal amplification (Self-priming hairpin-utilized isothermal amplification, SPHIA). The method for detecting a target nucleic acid using the SPHIA technology of the present invention overcomes the disadvantage of the conventional amplification method of an EXPAR technology that can detect only a target nucleic acid having a short length and a 3'-OH terminus as a target nucleic acid, and thus has the advantage that a target nucleic acid having a length of 500 mer or more through a positive feedback system of an amplification product can be detected with high detection efficiency.

Description

Target nucleic acid detection method using isothermal amplification technology using hairpin probe with self-primer structure

More particularly the invention relates to a method of detecting a target nucleic acid using an isothermal amplification technique (S elf- p riming airpin h-i sothermal utilized a mplification, SPHIA) utilizing a probe with hairpin primers rescuers, the nucleic acid The present invention relates to a hairpin probe having a self-priming structure for detecting a target nucleic acid that can be used for isothermal amplification and a method for detecting a target nucleic acid using the same.

Polymerase chain reaction (PCR) technology is the most widely used technology for amplification and detection of target nucleic acids. However, in order to implement the PCR reaction, precise temperature control is essential, and the volume of the PCR equipment is large and the price is high due to the mounting of a temperature control device for this purpose. Recently, as the demand for development of point-of-care testing (POCT) technology increases, interest in alternative technologies that can realize miniaturization by solving the disadvantages of PCR technology requiring a temperature control device is increasing.

In line with these technological trends, since the early 1990s, isothermal nucleic acid amplification techniques (nucleic acid sequence-based amplification (NASBA); helicase-dependent amplification (HDA); recombinase polymerase amplification ( RPA); strand displacement amplification (SDA); loop-mediated isothermal amplification (LAMP); rolling circle amplification (RCA); exponential amplification reaction (EXPAR)) have been actively developed (Van Ness et al., Proc. Natl. Acad). (Sci. USA , 100, 4504, 2003).

Among the above several isothermal nucleic acid amplification technologies, EXPAR technology has been regarded as a technology with high application potential as a POCT technology by realizing a target nucleic acid amplification efficiency of ^{up to 10 8 times within a short reaction time of about 30 minutes.} Specifically, the EXPAR technology is a hybridization reaction between a template and a target nucleic acid in which the cleavage enzyme recognition sequence (center of the template) and the target nucleic acid complementary nucleotide sequence (both ends of the template) are modified. It is a technology in which double-stranded DNA products are amplified exponentially. However, since the EXPAR technology amplifies a template using a target nucleic acid as a primer, there is a disadvantage that only a target nucleic acid having a short length and a 3'-OH end can be detected. has been of limited use.

Accordingly, the present inventors made diligent efforts to develop an isothermal amplification method having excellent detection efficiency regardless of the length of the target nucleic acid. In the case of detecting a target nucleic acid using a hairpin probe, it was confirmed that a target nucleic acid having a length of 500mer or more can be detected with high efficiency through a positive feedback system of an amplification product, and the present invention has been completed.

발명의 요약Summary of the invention

An object of the present invention is to provide a first hairpin probe having a self-priming structure for detecting a target nucleic acid.

Another object of the present invention is to provide a second hairpin probe that generates a target mimicking nucleic acid for detecting a target nucleic acid.

Another object of the present invention is to provide a method for detecting a target nucleic acid using the first hairpin probe having the self-primer structure and the second hairpin probe generating the target mimicking nucleic acid.

Another object of the present invention is to provide a composition for detecting a target nucleic acid comprising a first hairpin probe having the self-primer structure and a second hairpin pro generating the target mimicking nucleic acid.

Another object of the present invention is to provide a kit for detecting a target nucleic acid comprising a first hairpin probe having the self-primer structure and a second hairpin probe generating the target mimicking nucleic acid.

In order to achieve the above object, the present invention provides a first hairpin probe comprising the following components and having a self-priming structure for detecting a target nucleic acid:

(i) an X site located on the stem of a hairpin probe comprising a sequence complementary to a target nucleic acid;

(ii) an L site linked to the X site, having a sequence complementary to a target nucleic acid, and located in a loop of the hairpin probe;

(iii) the a site of the hairpin autologous hairpin primer, which is linked to the L site and includes a sequence complementary to a part of the X site to form the stem of the hairpin probe;

(iv) the b site of the autologous hairpin primer, which is linked to the a site of the autologous hairpin primer and includes a sequence complementary to a part of the X site to form the stem of the hairpin probe;

(v) the a' site of the autologous hairpin primer located at the 3' end of the hairpin probe, linked to the b site of the autologous hairpin primer, and comprising a sequence complementary to the a site of the autologous hairpin primer; and

(vi) an N site connected to the X site and having a cleavage enzyme recognition sequence; and

(vii) a T' region located at the 5' end of the hairpin probe, linked to the cleavage recognition domain, and comprising a sequence complementary to a trigger.

The present invention also provides a second hairpin probe comprising the following components and generating a target mimic nucleic acid for detection of a target nucleic acid:

(i) an S region located on the stem of a hairpin probe comprising a sequence complementary to a target nucleic acid;

(ii) an L2 region linked to the S region and positioned in a loop of the hairpin probe having a sequence complementary to the target nucleic acid;

(iii) an S' region linked to the L2 region and including a complementary sequence of the S region to form the stem of the hairpin probe;

(iv) an N site linked to the S' site and having a cleavage recognition sequence; and

(v) a T' region located at the 5' end of the hairpin probe, linked to the cleavage recognition domain, and comprising a sequence complementary to a trigger.

The present invention also provides a method for detecting a target nucleic acid comprising the steps of:

(a) reacting a composition comprising a target nucleic acid-containing sample, a first hairpin probe having the self-primer structure, a second hairpin probe generating the target mimicking nucleic acid, a nucleic acid polymerase, a cleavage enzyme, and a dNTP, to double-stranded DNA producing a product; and

(b) detecting the target nucleic acid by analyzing the generated double-stranded DNA product.

The present invention also provides a composition for detecting a target nucleic acid comprising a first hairpin probe having the self-primer structure, a second hairpin probe generating the target mimicking nucleic acid, a nucleic acid polymerase, a cleavage enzyme, and a dNTP.

The present invention also provides a kit for detecting a target nucleic acid comprising a first hairpin probe having the self-primer structure, a second hairpin probe generating the target mimicking nucleic acid, a nucleic acid polymerase, a cleavage enzyme, and a dNTP.

1 is a schematic diagram of the reaction of the present SPHIA technology. More specifically, (a) the trigger binding domain (T') and the cleavage enzyme recognition domain (N) are located at the 5' end of the hairpin probe 1, and the target nucleic acid complementary base sequence is located in the stem (X) and loop (L). The step of opening the hairpin probe 1 located at the 3' end of which the self-primer structure is modified by the hybridization reaction of the target nucleic acid; exposing the self-priming structure of the stem portion in the open hairpin probe so that the stem (a) and the 3' end portion (a') form an autonomous hairpin primer; (b) forming an intermediate product through polymerization by DNA polymerase in the self-hairpin primer of step (a); In this step, the target nucleic acid is separated and can be reused to open the hairpin probe 1 again; (c) generating a trigger (T) from the intermediate product of (b) by the combined action of a cleaving enzyme and a DNA polymerase; (d) binding the trigger (T) generated in the above step to the 3' end (T') of the hairpin probe 2; (e) generating a double-stranded DNA product (final product) as the trigger (T) is extended by a DNA polymerase using the hairpin probe 2 as a template; (f) a target mimic nucleic acid is generated by the combined action of a cleaving enzyme and a DNA polymerase, and the target mimic nucleic acid binds to the hairpin probe 1 again to promote reaction 1.

Figure 2 shows a schematic diagram of the positive feedback system of the present SPHIA technology. A positive feedback system is modeled in which the product of reaction 1 (trigger) promotes reaction 2, and the product of reaction 2 (target mimicking nucleic acid) promotes reaction 1. In Reaction 1, a process in which the target nucleic acid is removed and reused during the polymerization reaction by DNA polymerase is also included.

3 shows a schematic diagram of the hairpin probe 1 used in the SPHIA technology of the present invention. It is modeled after hybridization of hairpin probe 1 and target nucleic acid to form an autonomous hairpin primer. In the structure of hairpin probe 1, the trigger-complementary domain (T') and the cleavage enzyme recognition domain (N) are located at the 5' end, and the target nucleic acid complementary nucleotide sequence is located at the stem (X) and loop (L), , the 3' end consists of a self-priming structure. Since the hairpin probe is opened by the target nucleic acid, and the stem (a) and the 3' end portion (a') of the hairpin probe are composed of complementary sequences, a self hairpin primer structure is formed.

Figure 4 shows a schematic diagram of the hairpin probe 2 used in the SPHIA technology of the present invention. At the 3' end of hairpin probe 2, a trigger-complementary domain (T') and a cleavage enzyme recognition domain (N) are located, and the stem (S) and loop (L2) are composed of a target nucleic acid complementary nucleotide sequence. After the hairpin probe 2 and the trigger are hybridized, the double-stranded final product generated through polymerization by DNA polymerase contains the sequence of the target nucleic acid.

5 shows the experimental results performed to verify the effectiveness of the present SPHIA technology. More specifically, it shows the results of experimentally confirming whether a fluorescence signal is generated according to various conditions. In addition, the experimental results of confirming the amplification products generated under various conditions through electrophoresis are shown.

6 shows the test results of the target nucleic acid detection sensitivity of the present SPHIA technology (T _t : time to reach the threshold fluorescence signal value of the sample).

7 demonstrates the specificity of the present SHIA technology. It shows the results of the performance test for discriminating various base mismatches from the target nucleic acid. The D value is an index indicating the ability to discriminate base mismatches from the target nucleic acid, and may be characterized by being defined through the following relational expression.

D value = (T _t(Blank) - T _t(NT)) /(T _t(Blank) - T _t(Target) )

(T _t(NT) : time to reach the threshold fluorescence signal value of the sample containing the non-target nucleic acid; T _t(Blank) : the time to reach the threshold fluorescence signal value in the sample without the nucleic acid; T _t(Target) : the target nucleic acid time to reach the threshold fluorescence signal value of the sample containing

Figure 8 demonstrates the practical application of the present SPHIA technology. As a result of measuring the fluorescence signal using a nucleic acid having a long single-stranded structure obtained through asymmetric PCR as a sample, a signal value similar to that of a short nucleic acid sample was obtained.

Figure 9 demonstrates the RNA target applicability of the present SPHIA technology. As a result of the experiment after replacing the target nucleic acid with RNA of a single-stranded structure rather than DNA, the increased fluorescence signal was confirmed only in the sample containing the target RNA, and the formation of the amplification product was confirmed through electrophoresis. could

발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In the present invention, in order to develop a POCT (point-of-care testing) technology capable of detecting a target nucleic acid in the field, and to develop a technology capable of preempting market competitiveness, an isothermal cleavage and polymerization reaction system based on nucleic acid A new target nucleic acid detection method was developed through a nucleic acid amplification reaction.

The target nucleic acid detection method using the SPHIA technology provided in the present invention is an isothermal amplification technology that can solve the disadvantages of the target nucleic acid detection method represented by the conventional EXPAR technology. More specifically, in the present invention, in the presence of a target nucleic acid, the 3' end stem portion of hairpin probe 1 forms an autonomous hairpin primer, thereby causing an amplification reaction. The present invention is meaningful in that the amplification product generated during the amplification reaction promotes a positive feedback system, thereby realizing improved isothermal amplification efficiency. In addition, the present technology is significant in providing a technical method having excellent specificity for a target nucleic acid by introducing a target nucleic acid recognition system based on a hairpin structure.

Accordingly, in one aspect, the present invention relates to a first hairpin probe comprising the following components and having a self-primer structure for detecting a target nucleic acid:

(ii) an L site linked to the X site and positioned in a loop of the hairpin probe having a sequence complementary to the target nucleic acid;

In another aspect, the present invention relates to a second hairpin probe comprising the following components and generating a target mimic nucleic acid for detection of a target nucleic acid:

The structure of the first hairpin probe having the self-primer structure of the present invention is shown in FIG. 3 . The 5' end of the first hairpin probe of the present invention is composed of a sequence complementary to the trigger (T'), and the complementary trigger region is linked to the X site with a cleavage recognition site interposed therebetween. When a trigger site is synthesized by an autologous primer during hybridization of the target nucleic acid and the first hairpin probe, it is cleaved by a cleavage enzyme to generate a trigger (T), and the generated trigger (T) is the trigger complement of the second hairpin probe By binding to the site, the hairpin probe induces the production of a target mimic nucleic acid.

In the present invention, the L site of the first hairpin probe has a sequence complementary to the target nucleic acid positioned at the X site and a sequence complementary to the subsequent target nucleic acid. The L2 region of the second hairpin probe has a sequence complementary to the target nucleic acid positioned at the S region and a sequence complementary to the subsequent target nucleic acid.

In the present invention, when the target nucleic acid is present, reaction 1 in which an intermediate product and a trigger (T) are generated by elongation of the first hairpin probe proceeds, and the trigger (T) binds to the second hairpin probe and is polymerized Reaction 2 processes for generating a target mimic nucleic acid by an enzyme proceed (see FIG. 1 ).

(a) The trigger binding domain (T') and the cleavage enzyme recognition domain (N) are located at the 5' end of the hairpin probe 1, and the target nucleic acid complementary nucleotide sequence is located at the stem (X) and loop (L), 3 ' opening the hairpin probe 1 with a self-primer structure at the end by a hybridization reaction of the target nucleic acid; A step of exposing the self-priming structure of the stem portion in the open hairpin probe so that the stem (a) and the 3' end portion (a') form an autonomous hairpin primer (FIG. 1a);

(b) forming an intermediate product through polymerization by DNA polymerase in the self-hairpin primer of step (a); In this step, the target nucleic acid is separated and can be reused to open the hairpin probe 1 again (FIG. 1 b);

(c) generating a trigger (T) from the intermediate product of (b) by the complex action of a cleaving enzyme and a DNA polymerase (FIG. 1 c);

(d) binding the trigger (T) generated in the above step to the 3' end (T') of the hairpin probe 2 (FIG. 1 d);

(e) generating a double-stranded DNA product (final product) as the trigger (T) is extended by a DNA polymerase using the hairpin probe 2 as a template (FIG. 1e);

(f) A target mimic nucleic acid is generated by the combined action of a cleaving enzyme and a DNA polymerase, and the target mimic nucleic acid binds to hairpin probe 1 again to promote reaction 1 (FIG. 1 f).

In one aspect of the present invention, as a result of performing the SPHIA reaction according to the present invention using an analysis sample containing a target nucleic acid of various concentrations (100 aM ~ 1 nM), the detection limit of the target nucleic acid of the present invention (limit of detection, LOD) was 28.9 aM, and it was confirmed that the SPHIA technology proposed in the present invention has comparable performance to the existing EXPAR technology (FIG. 6).

In another aspect of the present invention, it was confirmed that, through SPHIA technology, not only can any nucleic acid sequence be discriminated, but also 1 to 3 nucleotide mismatches can be successfully discriminated (FIG. 7), through this , it was confirmed that the SPHIA technology proposed in the present invention has excellent specificity.

In another aspect of the present invention, in order to verify the practicality of the SPHIA technique of the present invention using the reaction conditions described in Example 1, a nucleic acid DNA of a long length single-stranded structure is obtained using asymmetric PCR, Validity was confirmed.

In another aspect of the present invention, various lengths and types of DNA (synthetic 59mer DNA, 221mer single-stranded DNA, 548mer single-stranded DNA) are prepared and used as a target nucleic acid to prepare a hairpin probe for the target nucleic acid. , it was confirmed that, when the SPHIA reaction was performed, it was possible to detect long-length nucleic acids longer than 500mer with similar efficiency to the short-length synthetic 59mer DNA.

In another aspect of the present invention, the SPHIA amplification reaction was performed by designing a hairpin probe using RNA of respiratory syncytial virus as the target nucleic acid. As a result, the increased fluorescence signal was confirmed only in the sample containing the target RNA, The formation of the amplification product was confirmed again through electrophoresis. Therefore, it was confirmed that the SPHIA technique according to the present invention can also detect RNA.

In another aspect, the present invention relates to a method for detecting a target nucleic acid comprising the steps of:

In the present invention, when the target nucleic acid is present, as described above, an intermediate is generated by elongation of the first hairpin probe having the self-primer structure, and a trigger is generated from the intermediate by a cleavage enzyme, and the generation After the trigger hybridizes with the trigger complementary region of the second hairpin probe that generates the target mimic nucleic acid, elongation occurs by a polymerase, and the target mimic nucleic acid and the trigger are generated by the cleavage enzyme. The trigger again binds to the second hairpin probe to perform hybridization and elongation, and the final product, a double-stranded DNA product, is generated by hybridization of the target mimic nucleic acids, and the double-stranded DNA product is analyzed to detect the target nucleic acid. can do.

The production of the double-stranded DNA product can be checked through electrophoresis, and can be detected using a fluorescent dye or other method capable of detecting dsDNA.

In the present invention, the target nucleic acid may be DNA, RNA, or DNA/RNA.

In one embodiment of the present invention, SYBR Green I was used as a fluorescent dye capable of detecting dsDNA, but the present invention is not limited thereto.

In another aspect, the present invention relates to a composition for detecting a target nucleic acid comprising the first hairpin probe having the self-primer structure, the second hairpin probe generating the target mimicking nucleic acid, a nucleic acid polymerase, a cleavage enzyme, and a dNTP .

In another aspect, the present invention relates to a kit for detecting a target nucleic acid comprising a first hairpin probe having the self-priming structure, a second hairpin probe generating the target mimicking nucleic acid, a nucleic acid polymerase, a cleavage enzyme, and a dNTP .

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Establishment of SPHIA technology reaction conditions

The sequences of the target nucleic acid and the first hairpin probe and the second hairpin probe used in this Example are shown in Table 1, and the SPHIA reaction solution preparation process is as follows.

SPHIA reaction solution (final 20 μL) was prepared by adding 0.4 μL of dNTPs (10 mM each), 5 μL of hairpin probe 1 (1 μM), 5 μL of hairpin probe 2 (100 nM), and 2 μL of various concentrations of target nucleic acids in reaction buffer solution. The reaction buffer was prepared by adding 1X CutSmart buffer (50 mM KAc (pH 7.9), 20 mM Tris-Ac, 10 mM Mg(Ac) ₂ , 100 μg/ml BSA) and 1X NEBuffer 2 (50 mM Tris) -HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT). Then, 0.5 µL of Klenow fragment (3'→5' exo-) (5 unit/µL) and 1 µL of Nt.alwi (10 unit/µL) were added to the reaction solution, and then SYBR at 37°C at 30 second intervals By measuring the fluorescence intensity generated from Green I, the final double-stranded DNA production amount was analyzed.

Example 2. Validation of SPHIA Technology

Using the same reaction conditions used in Example 1, various reaction conditions (1 to 4) were carried out validation experiments of the present technology.

(1) hairpin probe 1 + target nucleic acid + DNA polymerase + cleavage enzyme

(2) hairpin probe 1 + hairpin probe 2 + target nucleic acid + DNA polymerase

(3) hairpin probe 1 + hairpin probe 2 + target nucleic acid + DNA polymerase + cleavage enzyme

(4) hairpin probe 1 + hairpin probe 2 + DNA polymerase + cleavage enzyme

As a result of performing experiments according to various reaction conditions (1 to 4), as shown in a of FIG. 5, the reaction conditions ( In 3), it was confirmed that an extremely high fluorescence signal was generated.

In addition, through the validation experiment using electrophoresis, it was confirmed that a large amount of double-stranded DNA product was generated only under the reaction condition (3) (FIG. 5b).

Example 3. Sensitivity Verification of SPHIA Technology

A sensitivity verification experiment of the technique of the present invention was conducted using the reaction conditions mentioned in Example 1.

After preparing an assay sample (20 μL) containing a target nucleic acid of various concentrations (100 aM ~ 1 nM), the SPHIA reaction was performed. As shown in FIG. 6 , the detection limit of the target nucleic acid of the present invention of detection, LOD) was confirmed to be 28.9 aM. Through this experimental result, it was confirmed that the SPHIA technology proposed in the present invention has comparable performance to the existing EXPAR technology.

Example 4. Verification of target nucleic acid detection specificity of SPHIA technology

In order to confirm the target nucleic acid detection specificity of the SPHIA technology of the present invention, various base mismatch discrimination performance from the target nucleic acid was confirmed.

Nucleic acids other than the target nucleic acid The experiment was performed using any nucleic acid sequence and a sequence having 1 to 3 base mismatches with the target nucleic acid (Fig. 7b),

As a result, as shown in FIG. 7 , it was confirmed that, through the SPHIA technique, not only a random sequence of a nucleic acid could be distinguished, but also a mismatch of 1 to 3 nucleotides could be successfully discriminated.

The D value is an index indicating the ability to discriminate base mismatches from the target nucleic acid, and may be characterized by being defined through the following relational expression.

D value = (T _t(Blank) - T _t(NT)) /(T _t(Blank) - T _t(Target) )

Through this result, it was confirmed that the SPHIA technology proposed in the present invention has excellent specificity.

Example 5 Verification of Practicality of SPHIA Technology

In order to verify the practicality of the SPHIA technique of the present invention using the reaction conditions described in Example 1, a nucleic acid DNA having a long-length single-stranded structure was obtained using asymmetric PCR, and then the effectiveness of the SPHIA technique was confirmed.

Various lengths and types of DNA (synthetic 59mer DNA, 221mer single-stranded DNA, 548mer single-stranded DNA) were prepared, and using this as a target nucleic acid, hairpin probes for the target nucleic acid were prepared and the reaction was performed.

As a result, as shown in FIG. 8 , it was confirmed that long-length nucleic acids generated through asymmetric PCR were also detected with similar efficiency to short-length synthetic 59mer DNA.

Example 6. Verification of RNA target applicability of SPHIA technology

It was confirmed that target RNA can be detected using the SPHIA technique according to the present invention.

As the target nucleic acid, RNA of Respiratory syncytial virus was selected and a hairpin probe was designed to perform SPHIA amplification.

As a result, as shown in FIG. 9 , it was possible to confirm the increased fluorescence signal only in the sample containing the target RNA, and the formation of the amplification product was confirmed again through electrophoresis. Therefore, it was confirmed that the SPHIA technique according to the present invention can also detect RNA.

The target nucleic acid detection method using the SPHIA technology of the present invention solves the disadvantage that the existing EXPAR technology amplification method was capable of detecting only a target nucleic acid having a short length and a 3'-OH terminus as a target nucleic acid, and a positive feedback system for the amplification product It has the advantage of being able to detect target nucleic acids with a length of 500mer or more with high efficiency.

As a specific part of the present invention has been described in detail above, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

An electronic file is attached.

Claims

A first hairpin probe comprising the following components and having a self-priming structure for detecting a target nucleic acid:

(i) an X site located on the stem of the hairpin probe comprising a sequence complementary to the target nucleic acid;

(ii) an L site linked to the X site and positioned in a loop of the hairpin probe having a sequence complementary to the target nucleic acid;

(iii) the a site of the hairpin autologous hairpin primer, which is linked to the L site and includes a sequence complementary to a part of the X site to form the stem of the hairpin probe;

(iv) the b site of the autologous hairpin primer, which is linked to the a site of the autologous hairpin primer and includes a sequence complementary to a part of the X site to form the stem of the hairpin probe;

(v) the a' site of the autologous hairpin primer located at the 3' end of the hairpin probe, linked to the b site of the autologous hairpin primer, and comprising a sequence complementary to the a site of the autologous hairpin primer; and

(vi) an N site connected to the X site and having a cleavage enzyme recognition sequence; and

(vii) a T' region located at the 5' end of the hairpin probe, linked to the cleavage recognition domain, and comprising a sequence complementary to a trigger.
A second hairpin probe comprising the following components and generating a target mimicking nucleic acid for detection of the target nucleic acid:

(i) an S region located on the stem of a hairpin probe comprising a sequence complementary to a target nucleic acid;

(ii) an L2 region linked to the S region and positioned in a loop of the hairpin probe having a sequence complementary to the target nucleic acid;

(iii) an S' region linked to the L2 region and including a complementary sequence of the S region to form the stem of the hairpin probe;

(iv) an N site linked to the S' site and having a cleavage recognition sequence; and

(v) a T' region located at the 5' end of the hairpin probe, linked to the cleavage recognition domain, and comprising a sequence complementary to a trigger.
A method for detecting a target nucleic acid comprising the steps of:

(a) reacting a composition comprising a target nucleic acid-containing sample, a first hairpin probe having the self-primer structure of claim 1, a second hairpin probe generating the target mimic nucleic acid of claim 2, a nucleic acid polymerase, a cleavage enzyme, and a dNTP , generating a double-stranded DNA product; and

(b) detecting the target nucleic acid by analyzing the generated double-stranded DNA product.
The method of claim 3, wherein the target nucleic acid is selected from the group consisting of DNA, RNA and DNA/RNA.
The method according to claim 3, wherein the double-stranded DNA product is analyzed with a fluorescent signal material that recognizes double-stranded DNA.
A composition for detecting a target nucleic acid comprising the first hairpin probe having the self-primer structure of claim 1, the second hairpin probe generating the target mimic nucleic acid of claim 2, a nucleic acid polymerase, a cleavage enzyme, and a dNTP.
A kit for detecting a target nucleic acid comprising the first hairpin probe having the self-primer structure of claim 1 , the second hairpin probe of claim 2 , a nucleic acid polymerase, a cleavage enzyme, and a dNTP.