Hairpin probe combination for self-hybridization chain reaction, kit and application

Abstract

The invention provides a hairpin probe combination for a self-hybridization chain reaction, a kit and application, and belongs to the field of DNA amplification and detection. According to the self-hybridization chain reaction SHCR provided by the invention, a traditional HCR system which must contain two DNA hairpin probes is simplified into an SHCR system which only needs one DNA hairpin probe through simplified design by designing a palindromic sequence at the stem part of the DNA hairpin probe, so that the number of sequences in the reaction system is reduced, the design is simplified, the number of basic groups is also reduced, and the cost is reduced. Although the sequence number is reduced, the detection effect and capability are improved compared with those of the existing hybridization chain reaction HCR; the probe can also be used for detecting ATP concentration in cells, can be further applied to detection of cell survival and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present application relates to the field of DNA amplification and detection, and in particular, to a hairpin probe combination, a kit and an application thereof for self-hybridization chain reaction. Background Art

[0002] Hybridization chain reaction (HCR) was proposed by Pierce et al. in 2004. The system contains two DNA hairpin probes (H1 and H2). DNA hairpins are named because of their resemblance to hairpins. It generally consists of three parts: hairpin loop, hairpin stem and sticky end (or foothold). The complementary sequences between the two hairpin probes H1 and H2 are protected in the stems of their respective hairpin structures, so in the absence of a DNA trigger chain, H1 and H2 coexist metastably in the solution.

[0003] For traditional HCR, the number of bases of the DNA hairpin probe used in the system is generally 48 (including 6 hairpin loop bases, 6 sticky end bases and 36 hairpin stem bases). In order to reduce costs and the complexity of sequence design, people have simplified the HCR system and designed DNA hairpin probes with fewer bases (36-42 bases). However, the cost reduction brought about by this simplification is limited, and excessive reduction of the number of hairpin bases will lead to a significant increase in background signals. To solve this problem, Winssinger et al. reported a peptide nucleic acid (PNA)-based HCR system, which reduced the number of bases of the hairpin probe to 20 (including 5 hairpin loop bases, 5 sticky end bases and 10 hairpin stem bases). However, the PNA hairpin used for HCR needs to be modified with γ-polyethylene glycol to obtain the metastable state of the hairpin probe, HCR reaction performance and water solubility of the product. Asanuma et al. developed a HCR system based on left-handed acyclic D-threitol nucleic acid (D-aTNA). Since the D-aTNA double-stranded compound has extremely high thermal stability, the two hairpin probes can be reduced to 26 bases each (including 6 hairpin loop bases, 6 sticky end bases and 14 hairpin stem bases), ensuring that they can coexist in the system without causing high background signals.

[0004] The current HCR system structure is complex, resulting in complex operation; therefore, there is still a need to further simplify the HCR system. Summary of the invention

[0005] The current HCR system structure is complex and has many sequences. The system contains two DNA hairpin probes (H1 and H2). The DNA hairpin is named because of its resemblance to a hairpin or a hairpin. Figure 1). It generally consists of three parts: a hairpin loop, a hairpin stem, and a sticky end (or foothold). The complementary sequences between the two hairpin probes H1 and H2 are protected in the stems of their respective hairpin structures, so that in the absence of a DNA trigger chain, H1 and H2 coexist metastably in the solution.

[0006] Due to the complex structure and long sequence, the cost of sequence synthesis or reaction increases, resulting in an increase in the overall cost. In response to the current problem, the present application discloses a hairpin probe combination for self-hybridization chain reaction, which simplifies the structure and the sequence by designing a single hairpin probe chain and initiating the reaction with a trigger chain.

[0007] Hairpin probe combination of the present application In one aspect, the present application discloses a hairpin probe combination for self-hybridization chain reaction, the hairpin probe combination comprising a trigger strand and a hairpin probe; The hairpin probe comprises a first sticky end, a first complementary region, a hairpin loop, a second complementary region and a second sticky end which are sequentially linked by a phosphate bond; The first complementary region and the second complementary region are reverse complementary to form a hairpin stem; The first sticky end and the second sticky end form a sticky end.

[0008] In certain preferred embodiments, the hairpin stem sequence is a palindromic sequence; the number of cytosine bases and guanine bases in the hairpin stem sequence accounts for no less than two-thirds of the total number of bases; Preferably, the number of cytosine bases and guanine bases in the hairpin stem sequence accounts for two thirds of the total number of bases.

[0009] In certain preferred embodiments, the length of the first complementary region and the second complementary region is 18 bases.

[0010] In certain preferred embodiments, the sum of the number of bases of the first sticky end and the second sticky end is equal to the number of bases of the hairpin loop.

[0011] In certain preferred embodiments, the first sticky end and the second sticky end have the same sequence, and the first sticky end and the second sticky end are complementary to the hairpin loop, and the length of the hairpin loop is 12 bases.

[0012] In certain preferred embodiments, the trigger chain includes a first sticky end region, a complementary pairing region and a free segment, the first sticky end region and the free segment have the same sequence, and the first sticky end region is complementary to the first sticky end.

[0013] In certain preferred embodiments, the sequence of the hairpin probe is shown as SEQ ID NO.1, and the sequence of the trigger chain is shown as SEQ ID NO.2.

[0014] In the existing HCR system, when there is a DNA trigger chain, such as Figure 2 As shown, the sticky end (a) of the H1 hairpin complementarily hybridizes with the a* sequence in the trigger chain (I), opens the hairpin structure of H1, and generates I·H1. Then the newly exposed sequence c of H1 complementarily hybridizes with the sticky end (c*) of H2, opens the hairpin structure of H2, and generates I·H1·H2, so that the sequence (a*b*) in H2 that is exactly the same as that in the trigger chain is exposed, and then another H1 is opened, a cascade reaction occurs, and finally a long-chain DNA nanowire with a cut is generated.

[0015] In a specific embodiment of the present application, the first sticky end and the second sticky end of the hairpin stem in the hairpin probe are designed as a palindromic sequence, and the number of cytosine bases and guanine bases is limited to no less than two-thirds of the total number of bases; preferably, the number of cytosine bases and guanine bases of the hairpin stem sequence accounts for two-thirds of the total number of bases.

[0016] At the same time, the hairpin loop and the sticky end regions at both ends are designed to be complementary, such as the hairpin loop fragment is designed to be 12 adenine bases (A), and the two sticky end fragments are 6 thymine bases (T). This design can not only ensure the stability of the H hairpin structure, but also ensure that after I opens the hairpin structure H, the exposed sticky end can open another H hairpin structure, and the process is circulated to generate a long-chain DNA nanowire with a cut. Therefore, the probe combination designed in this application is named self-hybridization chain reaction based on a single hairpin (self-hybridization chain reaction), referred to as SHCR.

[0017] The reaction principle of the SHCR system based on the aforementioned single DNA hairpin probe is as follows: Figure 3 As shown. The symmetrical structural design of the hairpin structure H of the present application also allows the trigger chain I to open the hairpin structure H by binding to the 5' end or 3' end of the hairpin structure H, forming an intermediate product hairpin structure-trigger chain HI; subsequently, the opened hairpin structure H exposes a sequence that is completely consistent with the trigger chain I, which can open another hairpin structure H to repeat the chain reaction, and finally form a double-stranded DNA nanowire.

[0018] In another aspect, the present application provides a detection kit, which comprises any of the aforementioned hairpin probe combinations for self-hybridization chain reaction.

[0019] In certain preferred embodiments, it further comprises a buffer solution, wherein the buffer solution comprises: 20 mM tris(hydroxymethyl)aminomethane, 140 mM sodium chloride, 5 mM magnesium chloride, and a pH of 7.5.

[0020] The reaction system using buffer is as follows: the trigger chain and hairpin probe are diluted with buffer respectively, the concentration of hairpin probe during annealing is 500nM, the trigger chain and hairpin probe are annealed separately (maintained at 95°C for 5 minutes and slowly cooled to room temperature over 2 hours) to ensure the formation of hairpin structure. 500 nM hairpin probe and trigger chain of different concentrations are mixed in buffer solution to make the final concentration of hairpin probe 300 nM, and the resulting mixed solution can undergo SHCR reaction at room temperature.

[0021] In another aspect, the present application provides the aforementioned hairpin probe combination for self-hybridization chain reaction for use in detection.

[0022] Compared with the prior art, the beneficial effects of the present application include: the self-hybridization chain reaction SHCR (self-hybridization chain reaction) provided by the present application, through a simplified design, by designing a palindromic sequence at the stem of the DNA hairpin probe, simplifies the traditional HCR system that must include two DNA hairpin probes into a SHCR system that only requires one DNA hairpin probe, thereby reducing the number of sequences in the reaction system. The simplified design also reduces the number of bases. Although the number of sequences is reduced, the detection effect and ability are improved compared to the existing hybridization chain reaction HCR; it can also be used to detect intracellular ATP concentration, etc., and can also be further used to detect cell survival, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the structure of the DNA hairpin probe shown in this application; Figure 2 It is a reaction schematic diagram of the existing hybridization chain reaction HCR provided in this application; Figure 3 It is a schematic diagram of the structure and reaction principle of the self-hybridization chain reaction SHCR provided in this application; Figure 4 Schematic diagram of the hairpin structure envisioned by the designed self-hybridization chain reaction SHCR provided in Example 1; Figure 5 It is a schematic diagram of the principle of detecting ATP using the self-hybridization chain reaction SHCR provided in Example 3; Figure 6 is a schematic diagram of the analysis results of SHCR before and after the reaction using fluorescence spectroscopy, gel electrophoresis and atomic force microscopy imaging technology provided in Example 3; Figure 7 It is a schematic diagram of the results of comparing the detection effects of the hybridization chain reaction HCR and the self-hybridization chain reaction SHCR provided in Example 3; Figure 8It is a schematic diagram of the results of detecting ATP using the self-hybridization chain reaction SHCR provided in Example 3; Fig. 9 It is a schematic diagram of the principle results of detecting intracellular ATP using the self-hybridization chain reaction SHCR provided in Example 4; Fig.10 is a schematic diagram of evaluating cell survival by detecting ATP using the self-hybridization chain reaction SHCR provided in Example 5; Fig.11 The method for detecting UO2 by self-hybridization chain reaction (SHCR) provided in Example 6 2+ Schematic diagram of the principle; Fig.12 is a schematic diagram of the analysis results of SHCR before and after the reaction using gel electrophoresis and fluorescence spectroscopy provided in Example 6; Fig.13 is a schematic diagram of the analysis results of SHCR before and after the reaction using atomic force microscopy technology provided in Example 6; Fig.14 The method for detecting UO2 by self-hybridization chain reaction (SHCR) provided in Example 6 2+ Schematic diagram of the results; Fig.15 This is a schematic diagram of the results of metal ion specific detection using self-hybridization chain reaction SHCR provided in Example 6. DETAILED DESCRIPTION

[0024] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0025] The features and performance of the present application are further described in detail below in conjunction with the embodiments. Example 1

[0026] This embodiment designs a sequence of a self-hybridization chain reaction SHCR (self-hybridization chain reaction) based on a single hairpin.

[0027] In this example, adenosine triphosphate (ATP) is selected as a model molecule, and an aptamer that specifically binds to ATP is introduced to bind to the SHCR system. The designed hairpin probe combination includes a trigger chain and a hairpin probe, wherein the sequence of the hairpin probe is shown in SEQ ID NO.1, and the sequence of the trigger chain I is shown in SEQ ID NO.2.

[0028] By simulating the DNA sequence of the hairpin probe, the results are as follows Figure 4 As shown, the hairpin probe forms the expected hairpin structure ( Figure 4 A), the number of base pairings in the hairpin stem is 18, and the number of base pairings in the hairpin loop is 12. At the same time, the second structure obtained by simulation is a double hairpin structure ( Figure 4 B), which is caused by the introduction of the palindrome sequence. By simulating the thermodynamic properties of the two structures of the hairpin probe, it can be seen that although the Gibbs free energy change (ΔG) of the two structures is similar, the melting temperature (Tm) of the single hairpin structure is 89.7 ℃, which is significantly higher than the 74.1 ℃ of the double hairpin structure. This shows that the main stable structure formed by the hairpin probe during annealing is the single hairpin structure desired by the present invention. Example 2

[0029] Based on the clip-type probe combination designed in the experiment of Example 1, the fluorescent group FAM and the quenching group BHQ1 are modified on both sides of the hairpin stem of the hairpin probe, respectively, and the sequence of the hairpin probe with the fluorescent group and the quenching group is shown in SEQ ID NO.3; thereby, an ATP signal amplification biosensor based on the SHCR system is designed, and the fluorescence intensity is used as the signal output mode. Example 3

[0030] In order to compare the effects of the hairpin probe and the trigger chain of the hairpin probe combination designed in the present application, this example compares the hairpin probe combination of the present application with the traditional HCR.

[0031] In this example, adenosine triphosphate (ATP) was selected as a model molecule, and an aptamer that specifically binds to ATP was introduced to bind to the SHCR system.

[0032] A conventional HCR system with H1, H2 and trigger chain (I HCR ) has a similar sequence and structure to the SHCR system. In particular, H1 and H2 have the same sticky ends, hairpin loops and CG content of the hairpin stem as the hairpin probe of the present application. HCRIt has the same 6 A base design as the trigger chain I of the present application. These factors ensure the comparability between SHCR and HCR. In addition, the fluorophore (FAM) and the quencher group (BHQ1) are modified at the same position on the hairpin probe of the SHCR system and the H1 of the HCR system for fluorescence intensity detection. When the hairpin probe or H1 is in the hairpin form, the sufficiently close distance causes FAM to be quenched by BHQ1. When the hairpin is opened, the distance between FAM and BHQ1 increases, and the fluorescence signal is restored, proving the occurrence of a hybridization chain reaction. Considering that the FAM concentration in SHCR and HCR needs to be equal, half of the H in the SHCR system is not modified by FAM, that is, a naked hairpin probe, that is, SEQ ID NO.1.

[0033] The sequence of H1 in the HCR system is shown in SEQ ID NO.4, the sequence of H2 is shown in SEQ ID NO.5, HCR The sequence is shown in SEQ ID NO.6.

[0034] The modified hairpin probe and the unmodified hairpin probe were mixed to obtain the SHCR system, and H1 and H2 were mixed to obtain the HCR system; the final concentration of each type of DNA hairpin in the system was 250 nM, and after annealing according to the process in the above Example 1, the SHCR system was mixed with the trigger chain I, and the HCR system was mixed with I HCR Mix to ensure a final concentration of 150 nM for all hairpins and 50 nM for the trigger strand.

[0035] In addition, an ATP-binding aptamer was designed to combine with the SHCR system, and the fluorophore (FAM) and quencher group (BHQ1) were modified on both sides of the hairpin stem, thereby designing an ATP signal amplification biosensor based on the SHCR system, and using fluorescence intensity as the signal output method; in addition, a blocking chain was designed to block the reaction; the sequence of the aptamer trigger chain is shown in SEQ ID NO.7, and the sequence of the blocking chain is shown in SEQ ID NO.8.

[0036] The principle of SHCR system to detect ATP is as follows Figure 5 As shown in the figure, the designed aptamer trigger chain contains the trigger chain sequence and the ATP aptamer sequence, and the blocking chain BP partially hybridizes with the aptamer trigger chain, so that the aptamer trigger chain is partially blocked by the blocking chain BP and is not completely exposed. Therefore, in the absence of ATP, the SHCR reaction will not be triggered. Once ATP is present, the aptamer is recognized by ATP and binds to each other, exposing the complete trigger chain, thereby triggering the SHCR reaction.

[0037] The buffer solution used in the whole process of this system is: 20 mM tris(hydroxymethylaminomethane), 140 mM sodium chloride, 5 mM magnesium chloride, pH 7.5. The aptamer trigger chain (1 μM) and the blocking chain BP (3 μM) with a concentration of 3 times were mixed in the buffer solution, and the aptamer trigger chain-blocking chain BP complex was formed through an annealing process (maintained at 95°C for 5 minutes and slowly cooled to room temperature after 2 hours). The one-pot method was used to detect ATP, that is, the sample to be tested was mixed with the aptamer trigger chain-blocking chain BP and the hairpin probe in the buffer solution, and the final concentration of the hairpin probe was 300 nM and the aptamer trigger chain was 100 nM. Finally, incubate at room temperature for 3 h. According to the fluorescence characteristics of FAM, the excitation wavelength was set to 487 nm; the slit widths of excitation and emission were both set to 5.0 nm. Finally, the absolute fluorescence intensity of the reaction solution was detected by a fluorescence spectrophotometer.

[0038] In order to verify the feasibility of the SHCR system, the SHCR before and after the reaction was analyzed by fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. For fluorescence detection, we labeled the hairpin stem of the hairpin probe with a fluorophore (FAM) and a quencher (BHQ1). In the hairpin structure, the close distance between FAM and BHQ1 leads to fluorescence quenching, while the introduction of trigger chain I opens the hairpin structure, increasing the distance between the two and achieving the recovery of the fluorescence signal. Figure 6 As shown in A, the fluorescence signal of the SHCR system without I is weak, while the fluorescence signal of the system with I added is significantly enhanced, which indicates that during the reaction, the hairpin structure of the hairpin probe is opened, and FAM and BHQ1 move away from each other during the formation of DNA nanowires, so that the fluorescence signal of FAM is restored. In addition, Figure 6 The fluorescence signal change curve shown in B shows that the untriggered SHCR system exhibits a good metastable state, while the fluorescence intensity of the triggered SHCR system increases significantly and reaches a plateau after 3 h. We also characterized the SHCR system by gel electrophoresis. Figure 6 As shown in Figure 3, by introducing different concentrations of trigger chain I, DNA nanowires of different lengths can be clearly observed in the gel image, and the average molecular weight of the resulting DNA nanowires is inversely proportional to the concentration of trigger chain I, which is similar to traditional HCR. The AFM image also proves that after adding trigger chain I, the hairpin probe undergoes a self-hybridization chain reaction to generate DNA nanowires ( Figure 6 D).

[0039] In order to demonstrate the effectiveness and advantages of the SHCR system, we compared the SHCR system with the traditional HCR system by measuring the fluorescence signal curves of HCR and SHCR in response to time ( Figure 7A), and according to the signal-to-noise ratio calculation method reported by Jiang et al. (Jiang YS, Bhadra S, Li B, Ellington AD. Mismatches Improve thePerformance of Strand-Displacement Nucleic Acid Circuits [J]. AngewandteChemie International Edition. 2014;53(7):1845-1848), it was calculated that the signal-to-noise ratio of SHCR is about 36% higher than that of HCR ( Figure 7 B). In addition, compared with the traditional HCR system, the SHCR system exhibits higher single-base mismatch recognition ability ( Figure 7 C, Figure 7 D).

[0040] When ATP is used as a test object, Figure 8 As shown in A, the relative fluorescence intensity detected ( F / F 0 ,in F and F 0 The absolute fluorescence intensity corresponding to the presence or absence of ATP) is linearly related to the ATP concentration (y=0.01478x+1.01265, R2=0.998). The detection limit of ATP concentration calculated according to the calculation formula σ=3S / N (S is the standard deviation of the background fluorescence intensity, N is the linear slope) is 0.368 μM. At the same time, the SHCR system has a high selectivity for the detection of ATP ( Figure 8 B). Example 4

[0041] The SHCR system has also been used to detect intracellular ATP. The SHCR system is also used to detect intracellular ATP. The principle of the detection method is as follows: Fig. 9 As shown in A, the SHCR system was introduced into mouse breast cancer cells (4T1) by liposome transfection and incubated for 3 hours. Fig. 9 As shown in the CLSM fluorescence image B, the DNA probe was successfully transfected into 4T1 cancer cells and captured ATP in the cytoplasm to activate SHCR, producing a clear green fluorescence signal. This shows that the SHCR system has the ability to "turn on" fluorescence through ATP activation in cells. In addition, we further used the SHCR system to detect and distinguish different levels of ATP in cells. In order to achieve dynamic control of ATP expression, we used 5 mM Ca 2+4T1 cancer cells were pretreated with 300 nM oligomycin (which can inhibit ATP synthesis) for 30 min. Fig. 9 As shown in C, compared with untreated cells, Ca 2+ The treated cells showed significantly enhanced fluorescence, indicating that the dehydrogenases in the mitochondria were activated by Ca 2+ effectively activates and increases ATP production. Fig. 9 D shows that the fluorescence of cells treated with oligomycin is weaker, which is due to the reduced production of ATP in the cells. By quantifying the fluorescence signals of cells treated with different methods, it was found that Ca 2+ The fluorescence intensity of the treated 4T1 cancer cells was about two times higher than that of the untreated cells, while the 4T1 cancer cells treated with oligomycin showed only one-third of the fluorescence signal intensity of normal cells ( Fig. 9 E). In summary, in living cells, Ca can be detected by this SHCR system. 2+ Drugs such as oligomycin and oligomycin induce and inhibit changes in the amount of intracellular ATP. Example 5

[0042] The SHCR system can also be used to assess the survival of cells in culture. like Fig.10 As shown in A, since dead cells release ATP into the extracellular environment, the overall state of cells in the culture medium can be determined by introducing this system to detect the ATP content of the supernatant of 4T1 cells and human pancreatic adenocarcinoma (BxPC-3) cells. After irradiating the cells with X-rays (8 Gy), the SHCR system was introduced to measure the fluorescence intensity of the cell supernatant. The results are shown in Fig.10 As shown in B, the fluorescence intensity of cancer cells under X-ray irradiation increased nearly 4 times compared with untreated 4T1 cells and BxPC-3 cells, indicating that cell damage or death led to a large release of ATP from the cells. Example 6

[0043] In order to demonstrate that the SHCR system is still universal for other target molecules, this example selects uranyl ions (UO2 2 + ) as a model molecule, and introduced specific binding to UO2 2+ The 39E DNAzyme (39E DNAzyme) is combined with the SHCR system. The designed hairpin probe combination includes a DNA enzyme initiator chain-enzyme chain complex and a hairpin probe, wherein the sequence of the hairpin probe is shown in SEQ ID NO.1, the sequence of the initiator chain S is shown in SEQ ID NO.9, and the sequence of the enzyme chain E is shown in SEQ ID NO.10.

[0044] SHCR system detection of UO2 2+The principle is as follows Fig.11 As shown in the figure, the designed initiator chain S contains the SHCR trigger chain sequence and the substrate chain sequence, while the enzyme chain E partially hybridizes with the substrate chain to form 39E DNA enzyme, and at the same time hybridizes with the SHCR trigger chain, so that the trigger chain is partially blocked by the enzyme chain E and not completely exposed, playing a blocking role. 2+ In the presence of UO2 2+ When 39E DNA enzyme is 2+ It recognizes and breaks the trigger chain S at the adenine ribonucleotide (rA), freeing the trigger chain and exposing the complete trigger chain, thereby initiating the SHCR reaction.

[0045] The buffer solution used in the whole process of this system is: 50 mM 2-(N-morpholino)ethanesulfonic acid, 300 mM sodium chloride, pH 5.5. The DNA enzyme initiator chain S (1 μM) and the enzyme chain (2 μM) with a concentration of 2 times are mixed in the buffer solution and annealed (maintained at 95°C for 5 minutes and slowly cooled to room temperature over 2 hours) to form the DNA enzyme initiator chain-enzyme chain complex SE. Detection of UO2 2+ The one-pot method was used, that is, the sample to be tested was mixed with the DNA enzyme initiator chain-enzyme chain complex SE and the hairpin probe in a buffer solution, and the final concentration of the hairpin probe was 300 nM and the initiator chain was 200 nM. Finally, incubation was performed at room temperature for 50 min. According to the fluorescence characteristics of FAM, the excitation wavelength was set to 487 nm; the slit widths of excitation and emission were both set to 5.0 nm. Finally, the absolute fluorescence intensity of the reaction solution was detected by a fluorescence spectrophotometer.

[0046] In order to verify the feasibility of the SHCR system, the SHCR before and after the reaction was analyzed by gel electrophoresis, fluorescence spectroscopy and atomic force microscopy. Fig.12 As shown in A, we characterized the SHCR system by gel electrophoresis. 2+ Only a trace amount of DNA nanowires was produced in the system with only SE (lane 2). 2+ After that, it can be clearly seen that DNA nanowires of different lengths are generated (lane 3). In order to perform fluorescence detection, we labeled the hairpin stem of the hairpin probe with a fluorophore (FAM) and a quencher group (BHQ1). In the hairpin structure, the close distance between FAM and BHQ1 leads to fluorescence quenching, while the introduction of the trigger chain opens the hairpin structure, increasing the distance between the two and achieving the recovery of the fluorescence signal. Fig.12 As shown in B, does not contain UO2 2+ The fluorescence signal of the SHCR system is weak, while the addition of UO2 2+The fluorescence signal of the system was significantly enhanced, which indicates that during the reaction, the hairpin structure of the hairpin probe was opened, and FAM and BHQ1 moved away from each other during the formation of DNA nanowires, so that the fluorescence signal of FAM was restored. In addition, the AFM image also proved that the addition of UO2 2+ After that, a self-hybridization chain reaction occurs, from the point-like distribution of hairpin probes ( Fig.13 A) DNA nanowires transformed into linear structures ( Fig.13 B).

[0047] UO2 2+ When used as a test object for research, Fig.14 As shown, the relative fluorescence intensity detected ( F / F 0 ,in F and F 0 Corresponding to the presence or absence of UO2 2+ The absolute fluorescence intensity of UO2 2+ The concentration of UO2 2+ The concentration range was 0.05 nM to 20 nM and the relationship was linear (y=0.0398x+1.05, R 2 =0.998). UO2 was calculated according to the calculation formula σ=3S / N (S is the standard deviation of background fluorescence intensity, N is the linear slope). 2+ The concentration detection limit is 17.3 pM. 2+ The detection is highly selective ( Fig.15 ). Example 7

[0048] The SHCR system can also be used for UO2 in practical environments 2+ Concentration detection In order to verify the practical application of the SHCR system in real environment, we conducted a 2+ The tap water and Jinjiang River water were used as samples and spiked with different concentrations of UO2 2+ By substituting the above UO2 2+ Linear equation for concentration.

[0049] The specific results are shown in Table 1

[0050] The calculation results show that UO2 2+The recovery rate was between 99.85% and 105.75%, and the relative standard deviation (RSD) was between 3.94% and 9.89%, indicating that the SHCR system designed by the method provided in this application can achieve the detection purpose well.

[0051] In summary, the SHCR system provided in this application has a universal application effect and can be well applied to specific detection work.

[0052] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A hairpin probe assembly for self-hybridization chain reaction, characterized in that: The hairpin probe combination includes a trigger strand and a hairpin probe; The hairpin probe comprises a first sticky end, a first complementary region, a hairpin loop, a second complementary region and a second sticky end which are sequentially linked by phosphate bonds; The first complementary region and the second complementary region are reversely complementary to form a hairpin stem; The first sticky end and the second sticky end form a sticky end.

2. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The hairpin stem sequence is a palindromic sequence, and the number of cytosine bases and guanine bases in the hairpin stem sequence accounts for no less than two-thirds of the total number of bases; Preferably, the number of cytosine bases and guanine bases in the hairpin stem sequence accounts for two thirds of the total number of bases.

3. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The length of the first complementary region and the second complementary region is 18 bases.

4. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The sum of the base numbers of the first sticky end and the second sticky end is equal to the base number of the hairpin loop.

5. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The first sticky end and the second sticky end have the same sequence, and the first sticky end and the second sticky end are complementary to the hairpin loop, and the length of the hairpin loop is 12 bases.

6. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The trigger chain comprises a first sticky end region, a complementary pairing region and a free segment, the first sticky end region and the free segment have the same sequence, and the first sticky end region complementarily pairs with the first sticky end.

7. The hairpin probe assembly for self-hybridization chain reaction according to claim 1, characterized in that: The sequence of the hairpin probe is shown in SEQ ID NO.1, and the sequence of the trigger chain is shown in SEQ ID NO.

2.

8. A detection kit, characterized in that The detection kit comprises a hairpin probe combination for self-hybridization chain reaction as described in any one of claims 1 to 7.

9. The detection kit according to claim 8, characterized in that: The invention also comprises a buffer solution, wherein the buffer solution comprises: 20 mM tris(hydroxymethyl)aminomethane, 140 mM sodium chloride, 5 mM magnesium chloride, and the pH value is 7.

5.

10. Use of the hairpin probe combination for self-hybridization chain reaction according to any one of claims 1 to 8 in detection.

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