WO2010099662A1

WO2010099662A1 - A probe for nucleic acid real-time detection

Info

Publication number: WO2010099662A1
Application number: PCT/CN2009/070652
Authority: WO
Inventors: 何东华; 阮力; 郑立谋
Original assignee: 厦门艾德生物医药科技有限公司
Priority date: 2009-03-05
Filing date: 2009-03-05
Publication date: 2010-09-10

Abstract

A probe for nucleic acid real-time detection and the use thereof are provided. The probe includes: a single-stranded target sequence binding section complementary to the target sequence, a 5' terminal loop forming domain, and a 3' terminal loop forming domain, wherein the 5' terminal loop forming domain and the 3' terminal loop forming domain are respectively complementary to the sequence of target sequence binding section.

Description

A probe for real-time detection of nucleic acid

Technical field

The invention relates to the field of molecular detection, in particular to a novel probe for real-time detection of nucleic acid. The invention also relates to the use of dual loop probe technology.

Background technique

A variety of nucleic acid amplification techniques have been invented, especially the polymerase chain reaction (PCR) technique, which can specifically amplify target nucleic acid sequences to millions of times within 1.5 to 3 hours. In recent years, due to the advent of real-time PCR instruments, ordinary PCR eliminates the need for gel electrophoresis analysis, eliminating the need for post-PCR operations and eliminating PCR contamination. Therefore, real-time PCR technology is more and more widely used in clinical practice.

Real-time fluorescent PCR detection technology first used fluorescent dyes, such as SYBR GREEN, EVE GREEN, to indicate PCR reactions. Although the dye method is simple, it does not recognize non-specific amplification, especially due to non-specific amplification caused by primer dimers, and thus is greatly limited in practical applications. Subsequently, real-time fluorescent PCR detection technology began to apply labeled fluorescent nucleic acid probes, such as Taqman probes used in 5'-exonuclease technology, molecular beacons, fluorescent energy transfer probes (adjacent probes), and tweezers Primers, light probes, etc. The addition of the probe to the real-time PCR has a second recognition step for the PCR amplification product, thereby avoiding non-specific amplification and the result is more reliable. However, the probes described above generally have the disadvantages of complicated design, high fluorescence background, and limited ability to recognize single base mutations.

Summary of the invention

SUMMARY OF THE INVENTION The object of the present invention is to overcome the deficiencies of the prior art and to provide a novel probe for real-time detection of nucleic acids. The probe is fundamentally different in design from the current probe described above. This probe is simple in design, easy to synthesize, and has a low fluorescence background. The double loop probe detection technique of the present invention is based on direct hybridization of a target nucleic acid, which has two ring structures, 5' end and 3, since the 5' end and the 3' end both have a sequence complementary to the inside of the probe. The end-labeled fluorophore and quencher can sufficiently close and quench the fluorescence, so the fluorescence background is low during PCR annealing, and the two ring structures are also easy to open, so the hybridization efficiency to the target sequence is high.

The technical solution of the present invention is as follows:

The probe is an oligonucleotide, and the 5th and 3rd ends of the oligonucleotide are respectively 3-6 bases, and under certain conditions, the internal sequence of the probe can be double-stranded, and the probe is formed into 2 In the cyclic structure, the 5' end of the oligonucleotide is also labeled with a fluorescent group or a fluorescent donor, and the 3' end is labeled with a quencher or a fluorescent acceptor. In Under certain conditions, the probe moiety is double-stranded, forming two ring structures. When the probe hybridizes with the target sequence, part of the double-stranded structure is opened, the ring structure disappears, the probe is opened by the target sequence, and the fluorescence intensity changes. . Under appropriate conditions, the probe can bind to its fully complementary target sequence; as long as the target sequence contains a single base mutation, the probe can still retain a partial double-stranded and circular structure; or a probe designed to span The target sequence is variable, while maintaining a relatively high consensus hybridization efficiency for a variety of possible target sequences. According to this, the probe of the present invention can be used for detection of target nucleic acid in real-time PCR.

The nucleotide used in the probe of the present invention may be DNA, RNA, LNA (locked nucleic acid), or PNA (peptide nucleic acid), or any non-natural nucleoside composition.

Design method of double loop probe

Internal double-stranded binding region of the probe Tm value: In most cases, the complementary base of the probe is 6-20 bp, and its Tm value is equivalent to or higher than the annealing primer Tm value of 2-5 °C. According to the experimental results of a part of the specific embodiments of the present invention, the Tm value of the bicyclic probe complementary region of the real-time PCR for single-base mutation discrimination detection may be higher than the annealing temperature by 3-8 °C. For a shared probe having a multi-variable region target sequence, the Tm value of the double-stranded junction portion can be higher than the annealing temperature by 5-12 °C.

Probe length: In general, the total length of the probe is 12-80 bp, the target sequence binding region is 8-40 bp, the internal complementary region of the probe is the double-stranded binding region 6-20 bp, and the 5'-end loop-binding domain is 3-10 bp in length. The length of the 3-terminal loop-binding domain is also 3-10 bp.

Fluorescent labeling position: Both the fluorophore and the quencher can be labeled at the 5' or 3' end of the probe, but only one of the fluorophore and quencher can be labeled at one end.

Use of non-natural nucleotides: Any unnatural nucleotide, such as LNA, can be used at any position of the probe.

Optimal detection structure of double loop probes: Double loop probes can be used for amplification detection of specific target sequences in real-time PCR. The signal of the fluorophore is detected during the annealing phase. Current real-time PCR instruments for dual-ring probes include: Applied Biosystems (ABI) 7300, 7500, 7700, Bio-Rad's IQ Cycler, Roche's LightCycler 2.0, LightCycler 480, Corbett Research's Rotor-Gene 3000, 6000, Strategene's MX3000P and MX3005P and more.

Positive effect of double loop probe

Simple design: There are not many requirements for the design of the double-loop probe. The probe sequence is a part of the amplified target sequence, as long as the Tm value of the double-stranded binding region of the probe and the primer in the corresponding PCR reaction system or the annealing temperature used. The degree is consistent or higher. Anyone who has designed a PCR primer can design it.

Low fluorescence background: The fluorescent reporter group and the quenching group are very close, and the quenching effect is good.

Wide range of applications: For existing fluorescent probes, it has a good applicability to difficult templates in AT-rich and GC-rich regions.

Easy to design degenerate probes: Designed to ensure efficient specificity, it can be well indicated for multi-type templates with small regions of variable bases. Most of the existing probes are mostly It is possible to increase the number of probes matching the corresponding template to solve the problem, and the double-loop probe can solve the problem well by design. See Figure 2b.

Simple synthesis: no special instrument is required, just a normal DNA synthesizer can be used for synthesis. High specificity: Since the probe has its own reverse complementary double-stranded structure, only the binding of the target sequence to the probe binding region is greater than the binding energy of the reverse complementary duplex, and the probe can be generated with the target sequence. Hybridize, releasing a fluorescent signal. If there is a mutation in the target sequence, such as a single base change, the probe can maintain its own ring structure stability. In addition, due to the existence of a reverse complementary double-stranded structure, the length of the complementary region can be adjusted to adjust the specificity of the probe. DRAWINGS

Figure 1. Schematic diagram of a double loop probe and its reaction principle;

Figure 2a shows a schematic diagram of the reaction principle of the double loop probe in the PCR cycle detection;

Figure 2b, the double-loop probe hybridization reaction mechanism with the unmatched target sequence during PCR cycle detection; Figure 3, double-loop probe for real-time PCR detection; template serial 10-fold dilution;

Figure 4. Double loop probe SNP distinguishes hybridization ability;

Figure 5. Two double loop probes are used to detect single base mutations in real-time PCR;

Figure 6. Double loop probes are used in real-time PCR for the detection of variable target sequences. detailed description

Double loop probe

The bicyclic probe of the present invention is an oligonucleotide, and the 5' end and the 3' end of the oligonucleotide have 3-10 bases which can complement and bind to the inside of the probe, and under certain conditions, the two The sequence of the region can be double-stranded with the inside of the probe, allowing the probe to form two circular structures, and the 5' end of the oligonucleotide is also labeled with a fluorescent a light group or a fluorescent acceptor, the 3' end is labeled with a quencher or a fluorescent acceptor; the base forming the double-stranded region inside the probe can also be labeled with a fluorescent group or a fluorescent acceptor, or a quencher or Fluorescent receptor. Double-loop probes can have different morphology under different conditions, and the fluorescence value also changes. When the probe itself hybridizes to a partial double strand, forming two circular structures and a partially double-stranded structure, the fluorescent group and the quencher, or the fluorescent donor and acceptor, are in close proximity, and the fluorescent group or fluorescent donor is quenched. The quencher or fluorescent acceptor is quenched and the probe is not fluorescent in the wavelength range of the fluorophore or fluorescent donor reflection. When under denaturing conditions, such as acidic, basic or high temperature conditions, the double-stranded portion of the probe is opened, the ring structure disappears, the fluorophore or fluorescent donor is remote from the quencher or fluorescent acceptor, and the fluorophore or The fluorescent donor emits fluorescence. Under certain hybridization conditions, such as the annealing phase of PCR, the probe can bind to the target sequence in the reaction solution, the double-stranded portion of the probe is opened, the ring structure disappears, the fluorophore or fluorescent donor and the quencher or The fluorescent acceptor is far away, and the fluorescent group or fluorescent donor emits fluorescence.

Double loop probe reacts with target sequence

The bicyclic probe can react with a single stranded oligonucleotide in solution. The cyclic portion of the bicyclic probe and the internal double-stranded binding region of the probe can be combined with the target sequence to form a thermodynamically more stable duplex structure. The disappearance of the ring structure of the double loop probe causes the generation of fluorescence. A double loop probe can detect a perfectly matched target sequence in the reaction, see Figure 1.

According to Figure 1, the double loop probe consists of three different lengths of oligonucleotides 1, 2, 3. Region 1 consisting of 3-10 oligonucleotides at the 5' end is complementary to the internal base of the probe and can be double-stranded inside the probe, in this case referred to as the 5'-end loop-binding domain, 5' The end is labeled with a fluorescent agent 4; the region 3 composed of the last 3-10 oligonucleotides at the 3' end is complementary to the internal base of the probe, and can be double-stranded with the inside of the probe, which is referred to as 3' in this example. The end loops into the binding domain and the 3' end is labeled with a quencher 5; the probe also includes a longer intermediate region 2, which is complementary to the target sequence, in this case the target sequence binding site. The middle portion of the region 3 6-20 bases can be probed. The region 1 and the region 2 combine to form a double strand, so that the 5' end and the 3' end of the probe form a ring structure. When the 5' end and the 3' end are simultaneously looped, the probe does not fluoresce because the fluorescer is in close proximity to the quencher. After denaturation, the loop-forming structure is opened, and in the presence of the target DNA 6, the target sequence binding site binds to the target DNA, leaving the fluorescent agent and the quencher away. Thereby emitting fluorescence.

Double loop probes for nucleic acid amplification systems

As described above, the bicyclic probe of the present invention can hybridize with a single-stranded target sequence. From experiments, we further found that bicyclic probes can also be used to detect targets that are continuously exponentially amplified in a PCR system. Sequence. Three stages of high temperature denaturation, low temperature annealing and extension are included in each cycle of the PCR reaction. The double-loop probe generates fluorescence during the denaturation phase due to high temperature denaturation, so that the double strand cannot form. In the annealing stage, if no target sequence exists, the reverse complementary region of the probe will be combined into a double strand, and the probe forms a ring structure, and the fluorescent group It is close to the quencher without generating fluorescence; however, when a target sequence is present, the probe will hybridize to the target sequence, and the partial double strand formed by the probe itself will open, and the ring structure will disappear, thereby generating fluorescence. During the extension phase, the probe will dissociate from the target sequence. By detecting the fluorescent signal in the annealing phase of each cycle of the PCR, the amplified product can be detected in the real-time state. See Figure 2.

According to a double loop probe 1 1 and a double stranded amplification product 21 shown in Fig. 2, both are present in the PCR amplification reaction in the vector PCR cycle. The probe 11 comprises a 5'-end loop-binding domain chain 12, a target sequence junction link 13 and a 3'-end loop-binding domain chain 14, the 5' end is labeled with a fluorescent agent 15, and the 3' end is labeled with a quencher 16. Amplification product 21 comprises complementary strands 22, 23. At the time of high temperature denaturation, the 5' end ring and the 3' end ring of the dumbbell-shaped probe are opened, and the chains 22, 23 of the amplified product are separated. When the temperature is lower than the annealing temperature (for PCR primer annealing), the target sequence of the probe 11 binds to the target strand of the site 13 and its complementary amplification product. Fluorescent agent 15 is not quenched by the quencher and fluoresces.

As long as the number of complementary bases of the double-stranded binding portion of the probe itself and the number of bases in the circular region are adjusted, the ability of the probe to detect the mutated base or the unmatched base can be adjusted. For single-base mutations, the double-stranded binding portion must have a certain Tm value and the number of bases. The probe region 2 Tm value is about 2-5 °C higher than the Tm value of the double-stranded binding portion. For a shared probe possessing a multi-variable region target sequence, the Tm value of the double-stranded binding portion need not be too high, and is lower than the probe region 2 Tm value of 5-10 °C.

Example 1: Hepatitis B virus double loop probe real-time PCR detection

To investigate the effectiveness of dual-loop probe applications in real-time PCR detection, the PCR template was a series of diluted standard DNA samples. The total volume of the reaction system is 50 μl, including 5 μ 1 10 X buffer (160 mM [N ] ₂ S0 ₄ , 670 mM Tri s-HCl pH 8. 8, and 0. 1 % w/v Tween 20), 1 5 μ 1 25 mM MgCl ₂ , 400 μM per dNTP, upstream and downstream primers 0.4 μM, probe 0.1 μM, 1. 0 U 73⁄4 g DNA polymerase, 5 μl template DNA. Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine. The reaction conditions were: pre-denaturation at 96 ° C for 2 min, followed by denaturation at 96 ° C for 15 s, 68 ° C _59 ° C (1 ° C temperature drop per cycle) 15 s, 72 ° C extension for 15 s, 10 cycles; 94 °C 3 min; 94 °C 15 s, 58 °C 20 s (test FAM, fluorescent signal), 72 °C 15 s, a total of 35 cycles. Standard DNA was serially diluted 10-fold and 0 was used as a negative control. The bicyclic probe comprises a nucleic acid sequence that is complementary to the amplification product. On The downstream primer amplified the S region gene of the hepatitis B virus (NC-003977) and amplified 174 bp. The upstream primer is: 5'-caacatcaggattcctaggacc-3', and the downstream bow is 5 '-ggtgagtgattggaggttg-3 '. The target sequence of the probe is close to the upstream primer. The sequence is: FAM-5'- AGTCTAG

CAGAGT|CTAGACTCGTGGT|GGACTTCACCACG- 3 '-BHQ. The bases indicated by the boxes on the probes are the self-double-stranded binding regions within the probe, and the bases underlined at the 5 and 3 ends are the bases involved in the formation of the double-stranded binding region within the probe.

Fluorescence real-time detection measured 35 cycles during PCR amplification, and the results are shown in Fig. 4. The initial target concentration is at line 41 when the dilution is most concentrated, and lines 42, 43, and 44 respectively indicate fluorescence curves after the template has been diluted 10 times for 3 consecutive gradients. Line 45 illustrates the comparison of no targets (water).

At the same concentration as the primer or 1/2 to 1/4 of the primer concentration, the bicyclic probe can be rapidly hybridized to the target sequence to display a fluorescent signal. In the absence of the target sequence, the complementary base of the probe hybridizes intramolecularly to form a stable bicyclic structure and a double-stranded structure, the fluorophore and the quenching group are close together, and the autofluorescence is quenched. Therefore, the double loop probe can be used for real-time nucleic acid amplification detection and can be used in PCR detection technology for detecting hepatitis B virus.

Example 2: Double loop probe for distinguishing single base mutant target sequence The HPA-1 gene of human platelet al loantigen (HPA) was selected as a research object. HPA-1 results in two antigens, HPA-la and HPA-lb, due to a single base mutation, the T>C mutation. The double loop probe is designed to target the HPA-la antigen gene, and the sequence is: 5 ' -HEX-TGG

|GCTC^GGTGAGCCCAGAGGCAGG|TGCCTC|-Dabcyl-3 ' , the underlined base is a mutation-generating base, and the outer-framed base is a loop-forming binding base. The synthesized hybridization sequence is 2 bases more at each end than the probe. The HPA-la target sequence is: 5'-GCCCTGCCTCIGGGCTCACCTCG-3', the underlined base is the mutation generating base; the HPA-lb target sequence is: 5'-GCCCTGCCTC GGGCTCACCTCG-3', the underlined base is The base of the mutation occurs; using a 25 μΐ reaction system containing IX buffer ( 67 mM Tris-HCl , 16. 6 mM (NH ₄ ) ₂ S0 ₄ , 85 g/mL BSA, pH 8. 0), 2. 0 mM MgCl ₂ , 0.4 μ μ probe and 1. 6 μΜ hybridization sequence. The reaction procedure was: 94 ° C for 1 min; 94 ° C for 10 s, 56 ° C for 15 s, 40 cycles. HEX fluorescence data was acquired at each hybridization (i.e., at 56 °C) using a Rotor-Gene 3000 real-time PCR machine.

Figure 4 shows real-time fluorescence of a fully complementary target sequence (line 31) and a mutated target sequence (line 32) Light signal (Fluoresoenoe) observation. It can be seen that the fluorescence intensity has increased by more than 30 times. If there is a single nucleotide or more than one nucleotide mutation in the target sequence, no fluorescent signal will be produced. Example 3: Detection of SNP by double-loop probe in real-time PCR The HPA-4 gene of human platelet alloantigen (HPA) was selected, and a G>A mutation was present in the gene, resulting in HPA- 4a and HPA-4b two platelet antigens, HPA-4a is wild type, and HPA-4b is mutant type. Two-loop probes were designed for the gene sequences of the two antigens. The two probes differed by only one base, and three typical samples of known genotypes were selected for testing. One was a pure and HPA-4a sample, one was a pure and HPA-4b sample, and one was a hybrid sample containing both HPA-4a and HPA-4b. At the same time, a no (negative) sample, 0 control, was made during the test. The wild-type (HPA-4a) probe sequence is: 5'-FAM-CGAAAGCTGGTGAGCTTTCGCATCTGGGTCCAGATG-BHQ-3', and the mutant (HPA-4b) probe sequence is:

5, -HEX -BHQ-3', the underlined base is a mutation-generating base, and the outer-framed base is a loop-forming binding base. The upstream primer is:

5' -GGACCTGTCTTACTCCATGAAGG-3 ' ; The downstream primer is:

5' -GAAGCCAATCCGCAGGTTAC- 3'.

The total reaction system is 25 μL, including 2.5 μ 1 10 X buffer (160 mM [NH ₄ ] ₂ S0 ₄ , 670 mM Tris-HCl pH 8.8, and 0.1% w/v Tween 20), 1.5 μ 1 25 mM MgCl ₂ , 400 μM per dNTP, 0.4 μM per primer, 0.1 μμ per probe, 1.0 U 73⁄4 g DNA polymerase, 20 ng template DNA. Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine. The reaction conditions are: pre-denaturation at 96 °C for 2 min, followed by denaturation at 96 ° C for 15 s, 68 ° C _59 ° C (temperature drop of 1 ° C per cycle) 15 s, 72 ° C extension for 15 s, 10 cycles; 94 ° C 3 min; 94 °C 15 s, 58 °C 20 s (detecting FAM and HEX fluorescence signals), 72 °C for 15 s, a total of 35 cycles.

Figure 5 shows the results of real-time fluorescent PCR cycle data. The fluorescence emitted by the probe corresponding to the wild-type target is solidly represented by the square, and the fluorescence emitted by the probe corresponding to the mutant heterologous target is represented by a solid ring.

The wild-type probe (curve 51) and the mutant probe (curve 52) did not fluoresce relative to the negative sample. The results show that the corresponding fluorescence intensity is enhanced only when the template is included in the reaction. When both targets are in the sample, the wild type probe (curve 57), and the mutant probe (curve 58), the fluorescence intensity increases significantly, However, for wild-type targets, the fluorescence intensity was significantly enhanced from the wild-type probe (curve 55) rather than the mutant heterologous probe (curve 56). In contrast, for mutant targets, the fluorescence intensity was significantly enhanced from the mutant probe (curve 53) rather than the wild-type probe (curve 54). The results show that only matching probes can produce the correct signal. 100% complete detection of wild template and mutant heterologous template. This demonstrates that the probe according to the invention distinguishes the target by a single nucleoside. No signal is found when there is no template, and when there are two templates, both signals are found.

Implementation column 4: Double loop probes are used as degenerate probes to detect multivariate target sequences

In the clinical application of real-time PCR, especially the detection of pathogenic microorganisms in infectious diseases, there are often many types of pathogenic microorganisms, and the mutation is fast. Even the conserved genes of the pathogenic microorganisms can find each type. The same sequence, used for primer and probe design, often requires multiple detection probes for multiple types, which not only increases the cost, but also increases the difficulty of the experimental design. This example uses a double loop probe for real-time PCR detection of Ureaplasma urealyticum (UU). Ureaplasma urealyticum can be divided into 14 standard serotypes. Amplification primers and probes were designed using the region of the urease gene that is more conserved in Ureaplasma urealyticum. The upstream primers are: 5, - GATCACATTTCCACTTATTTGAAACA-3, ; The downstream primers are:

5 '-AAACGACGTCCATAAGCAACTTTA-3 '. The probe is: FAM-5 ' - CAGGTGC

ACCACAAGCACCTG CTACGATTTTGTTC^AATCGTAG -3 '-BHQ The underlined base is a polybasic base, and the outer base is a loop-forming base.

The total reaction system is 25 μL, including 2. 5 μ 1 10 X buffer (160 mM [NH ₄ ] ₂ S0 ₄ , 670 mM Tris-HCl pH 8. 8, and 0. 1 % w/v Tween 20), 1. 5 μl 25 mM MgCl ₂ , 400 μM per dNTP, 0.4 μμ of each primer, 0.1 μ μ of each probe, 1. 0 U 73⁄4 g DNA polymerase, 20 ng of template DNA. Real-time PCR reactions were performed on a RotorGene 3000 real-time PCR machine. The reaction conditions are: pre-denaturation at 96 ° C for 2 min, followed by denaturation at 96 ° C for 15 s, 68 ° C _59 ° C (1 ° C temperature drop per cycle) 15 s, 72 ° C extension for 15 s, 10 cycles; 94 ° C 3 min; 94 °C 15 s, 58 °C 20 s (detecting FAM fluorescence signal), 72 °C 15 s, a total of 35 cycles.

Figure 6 shows the results of real-time fluorescent PCR cycle data. A, B, C, D, E, and F represent the detection results of different Ureaplasma urealytic serotype samples, and the probes are well detected regardless of the serotype.

Industrial applicability

The invention relates to a probe for real-time detection of nucleic acid, because the probe has its own reverse complementation The double-stranded structure, therefore, the probe of the present invention is highly specific; in addition, due to the existence of a reverse complementary double-stranded structure, the length of the complementary region can be adjusted during design to adjust the specificity of the probe. Therefore, the present invention has good industrial applicability.

Claims

Rights request

A probe for real-time detection of nucleic acid, comprising:

a single-stranded target sequence binding site that is complementary to the target sequence;

a 5' end of the 5' end of the binding site of the single-stranded target sequence into a loop-binding domain;

a 3' end of the 3' end of the binding site of the single-stranded target sequence into a loop-binding domain;

Wherein the 5'-end loop-binding domain and the 3'-end loop-binding domain are complementary to a sequence in the binding site of the target sequence, respectively, so that the probe becomes two loop structures;

One end of the 5' or 3' end of the probe is labeled with a fluorescent group or a fluorescent donor, and the other end is labeled with a quencher or a fluorescent acceptor.

The probe for real-time detection of nucleic acid according to claim 1, wherein the 5'-end loop-binding domain is 3-10 bases in length.

The probe for real-time detection of nucleic acid according to claim 1, wherein the 3'-end loop-binding domain is 3-10 bases in length.

The probe for real-time detection of nucleic acid according to claim 1, wherein the probe has a total length of 12-80 bp, wherein the target sequence binding region is 8-40 bp.

The probe for real-time detection of nucleic acid according to claim 1, wherein the nucleotide in the probe is DNA or RNA.

The probe for real-time detection of nucleic acid according to claim 1, wherein: the target sequence binding site, the 5' end loop-binding domain and the 3' end loop-binding domain, at least One includes at least one unnatural nucleoside.

A method for amplifying and detecting a real-time nucleic acid target sequence, wherein the target amplification product is detected by a fluorescently labeled probe, comprising performing hybridization detection on the target sequence using a single-stranded nucleic acid probe, comprising: Complementary single-stranded target sequence binding site;

a 5' end looping binding domain located at the 5' end of the binding site of the single-stranded target sequence; and a loop-binding domain at the 3' end of the 3' end of the binding site of the single-stranded target sequence;

The method for amplifying and detecting a real-time nucleic acid target sequence according to claim 7, wherein: The 5' end loop-forming domain is 3-10 bases in length.

9. A method for amplifying and detecting a real-time nucleic acid target sequence according to claim 7, wherein the 3'-end loop-binding domain is 3-10 bases in length.

10. A method for amplifying and detecting a real-time nucleic acid target sequence according to claim 7, wherein: said probe

The total length is 12-80 bp, and the target sequence binding region is 8-40 bp.

The method for amplifying and detecting a real-time nucleic acid target sequence according to claim 7, wherein the nucleotide in the probe is DNA or RNA.

The method for amplifying and detecting a real-time nucleic acid target sequence according to claim 7, wherein: the target sequence binding site, the 5'-end loop-binding domain, and the 3'-end loop-binding domain, At least one includes at least one unnatural nucleoside.

13. A probe for real-time detection of nucleic acid, characterized in that: the probe is an oligonucleotide, and the 5 and 3 ends of the oligonucleotide are respectively 3-6 bases, under suitable conditions. It can be double-stranded with the internal sequence of the probe, and the probe forms two circular structures. One end of the 5' end or the 3' end of the oligonucleotide is labeled with a fluorescent group or a fluorescent donor, and the 5' end or The other end of the 3' end is labeled with a quencher or a fluorescent acceptor; when the probe hybridizes to the target sequence, part of the double-stranded structure is opened, the circular structure disappears, the probe is opened by the target sequence, and the fluorescence intensity changes.

14. A method of detecting a nucleic acid target sequence, comprising: adding a probe according to any one of claims 1 to 6 or 13 to a sample suspected of having a target sequence, and measuring the indication with said target Sequence hybridization of fluorescently labeled fluorescence increases.