Multifunctional dumbbell-shaped nucleic acid probe, kit, target detection method and application of multifunctional dumbbell-shaped nucleic acid probe

Abstract

The invention relates to the field of nucleic acid detection, in particular to a multifunctional dumbbell type nucleic acid probe, a kit, a target detection method and application thereof. The multifunctional dumbbell type nucleic acid probe provided by the invention comprises two or more of the following functional areas: a promoter area used for being combined with RNA polymerase; the target recognition area is used for recognizing and combining a target sequence and opening the dumbbell type nucleic acid probe; the dumbbell type nucleic acid probe can be transcribed and combined with a rolling ring, a specific sequence is subjected to mass transcription, amplification of the specific sequence is achieved, finally, signal amplification is achieved during detection, and the dumbbell type nucleic acid probe has the advantages of being simple in reaction system, low in background signal and high in detection specificity; and the dumbbell type nucleic acid probe can be designed according to different target sequences, so that the controllable detection of different targets can be realized.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection, specifically to a multifunctional dumbbell-shaped nucleic acid probe, reagent kit, target detection method, and their applications. Background Technology

[0002] Multifunctional dumbbell-shaped DNA, due to its conformation and function, can be rationally designed to accurately identify targets and exert catalytic effects. It can be used as a recognition element, recognizing a wide range of analytes from metal ions to microorganisms. Dumbbell-shaped DNA probes can be flexibly designed to be combined with different amplification methods, improving the detection performance and accuracy of biosensor platforms. Furthermore, they can be designed as molecular switches combined with logic gate strategies to detect pathogenic microorganisms or small molecule targets, making their applications quite broad. In addition, dumbbell-shaped DNA has natural structural advantages and extremely high stability, avoiding non-specific amplification during rolling circle transcription amplification, thus improving the detection performance of the detection system. Moreover, dumbbell-shaped functional nucleic acids have high programmability and can be used as recognition probes or signal probes, with a wide range of potential applications.

[0003] To further improve sensor sensitivity, various amplification methods are commonly employed, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), chain shift amplification (SDA), nucleic acid sequence amplification (NASBA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA). Among these, RCA is favored by many researchers due to its advantages of high amplification efficiency, low error rate, and simple operation.

[0004] Rolling circle transcription (RCT), a technique in isothermal amplification, was established in 1995 by Eric T. Kool's team by mimicking the rolling circle replication process in viroids. It achieves cyclic catalysis of small circular oligonucleotide substrates to obtain long single-stranded RNA with periodic repetition. RCT has a higher amplification yield than linear radial runoff transcription, at least 450-fold, and a shorter amplification time, transcribing long single-stranded RNA of approximately 500–7500 nt, corresponding to 6–90 rotations of an 80 nt circle, taking less than 1 hour. RCT can be classified into linear RCT, multi-primer RCT, and exponential RCT. Furthermore, due to the programmability of the circle, the ribozyme encoding the RCT product can be trans-cleaved to control the long single-stranded RNA to a pre-designed length, which facilitates the catalytic release of functional RNA monomers.

[0005] Currently, biosensing research combining isothermal amplification technology and CRISPR has made some progress. For example, existing studies have reported the detection of microRNA based on DNA dumbbell probes, rolling loop transcription, and CRISPR-Cas12a binding. However, this technology suffers from problems such as complex reaction systems, poor stability, high background signals, and low detection specificity.

[0006] However, no existing technology has proposed a DNA dumbbell probe, rolling loop transcription, and CRISPR-Cas binding detection method that has the advantages of simple reaction system, low background signal, and high detection specificity. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a multifunctional dumbbell-shaped nucleic acid probe, reagent kit and target detection method and its uses. The detection method has the advantages of simple reaction system, low background signal and high detection specificity, and can controllably detect different targets.

[0008] Therefore, the present invention provides the following technical solution:

[0009] The embodiments disclosed in this invention relate to dumbbell-shaped nucleic acid probes, including the following functional regions:

[0010] The promoter region is used to bind to RNA polymerase.

[0011] The target recognition region is used to identify and bind to the target sequence and open the dumbbell-shaped nucleic acid probe.

[0012] As an alternative implementation, it further includes a linker region for linking to form a circular template. As an alternative implementation, the promoter region includes an RNA polymerase promoter region.

[0013] As an alternative implementation, the promoter region is located upstream of the polymerization direction of RNA polymerase in the dumbbell-shaped nucleic acid probe;

[0014] In a preferred embodiment, when the promoter region is a T7 promoter region, its position is upstream of the T7 RNA polymerase polymerization direction 5'→3', located near the 5' end in the dumbbell-shaped nucleic acid probe.

[0015] As an alternative implementation, the connection region is located at the 5' and 3' ends of the dumbbell-shaped nucleic acid probe.

[0016] As an alternative implementation, the functional region further includes an amplification functional region for integration with nucleic acid amplification technology to achieve signal generation and amplification. In some implementations, the amplification functional region can be combined with amplification technologies, such as common isothermal amplification technologies like loop-mediated isothermal amplification (LAMP), nick endonuclease isothermal amplification (NERA), sequence-dependent amplification (NASBA), rolling circle amplification (RCA), helicase-dependent DNA isothermal amplification (HDA), recombinase polymerase amplification (RPA), and enzyme-catalyzed recombination isothermal amplification (ERA).

[0017] In a preferred embodiment, the amplified functional region further includes a crRNA direct repeat sequence transcription complementation region, used for rolling circle transcription to generate crRNA direct repeat sequences. In the long single-stranded RNA generated after rolling circle transcription, the crRNA direct repeat sequence and its upstream or downstream sequence (as a spacer sequence) form crRNA.

[0018] As an alternative implementation, the functional regions on the single-stranded nucleotide sequence of the dumbbell-shaped nucleic acid probe are sequentially arranged along the 5'→3' direction, and self-assembled to form the dumbbell-shaped nucleic acid probe according to the base complementary pairing principle; or

[0019] The dumbbell-shaped nucleic acid probe is a DNA sequence; or

[0020] The 5' end of the dumbbell-shaped nucleic acid probe is phosphorylated.

[0021] As an alternative implementation, the dumbbell-shaped nucleic acid probe has the following characteristics: ΔG < 0; Tm: 10-90℃. Further, Tm can be any value between 10, 20, 30, 40, 50, 60, 70, 80, and 90℃, or a range between any two values. Under these conditions, it can be ensured that the single-stranded nucleotide sequence of the designed dumbbell-shaped nucleic acid probe self-assembles into a dumbbell-shaped nucleic acid probe through the base complementary pairing principle. Therefore, different dumbbell-shaped nucleic acid probes can be designed for different target sequences based on the above design principles. For example, the single-stranded nucleotide sequences of the designed dumbbell-shaped nucleic acid probes for detecting miR-21 and Staphylococcus aureus are shown in Table 1.

[0022] As a preferred embodiment, at least one of the following probes is included:

[0023] Probe 1, from the 5' end to the 3' end, includes a linker region I, a T7 promoter region, a target recognition region, a crRNA direct repeat sequence transcription complementation region, and a linker region II; the linker region I and the linker region II partially complement each other with the target recognition region to form a stem region, and the T7 promoter region and the crRNA direct repeat sequence transcription complementation region form a loop region;

[0024] Probe 2, from its 5' end to its 3' end, comprises a target recognition region I, a T7 promoter region, an activation sequence complementary hybridization region, a crRNA direct repeat sequence transcription complementary region, and a target recognition region II. The target recognition region I partially hybridizes with the T7 promoter region to form a stem region, the activation sequence complementary hybridization region partially hybridizes with the target recognition region II to form a stem region, and a portion of the T7 promoter region and the crRNA direct repeat sequence transcription complementary region each form a circular region. Optionally, the length of the activation sequence complementary hybridization region is ≥21 nt and ≤25 nt.

[0025] Probe 3, from its 5' end to its 3' end, comprises a linker region I, a target recognition region, a T7 promoter region, a crRNA direct repeat sequence transcription complementation region, and a linker region II. The linker region I and the target recognition region partially complement each other to form a stem region, the linker region II and the T7 promoter region partially complement each other to form a stem region, the target recognition region forms a stem-loop region, and the crRNA direct repeat sequence transcription complementation region forms a loop region.

[0026] Probe 4, from the 5' end to the 3' end, includes a linker region I, a crRNA direct repeat sequence transcription complementary region, a target recognition region, a T7 promoter region, and a linker region II; the linker region I and the linker region II respectively partially complement each other with the target recognition region to form a stem region, and the crRNA direct repeat sequence transcription complementary region and the T7 promoter region respectively form a loop region.

[0027] In some embodiments, the nucleotide sequence of the T7 promoter region is as shown in positions 11 to 30 of SEQ ID NO.1;

[0028] And / or, the nucleotide sequence of the transcribed complementary region of the crRNA direct repeat sequence is as shown in positions 52-72 of SEQ ID NO.1.

[0029] In some embodiments, the nucleotide sequence of probe 1 is shown in SEQ ID NO.1;

[0030] The nucleotide sequence of probe 2 is shown in SEQ ID NO.2;

[0031] The nucleotide sequence of probe 3 is shown in SEQ ID NO.3;

[0032] The nucleotide sequence of probe 4 is shown in SEQ ID NO.4.

[0033] The embodiments disclosed in this invention relate to a target detection kit based on a dumbbell-shaped nucleic acid probe, comprising:

[0034] The dumbbell-shaped nucleic acid probe, ligase, RNA polymerase, activation sequence, buffer, NTP and / or reporter gene are described.

[0035] In some embodiments, a Cas protein is included, wherein the Cas protein is selected from Cas12a, Cas13a, Cas9, or Cas14;

[0036] In some embodiments, the activation sequence can be recognized by a sequence upstream of the transcription complementary region of the crRNA direct repeat sequence, or optionally by a target recognition region, an activation sequence complementary hybridization region, or a promoter region; optionally, the nucleotide sequences of the activation sequences corresponding to probes 1 to 4 are as shown in SEQ ID NO. 5 to 8, respectively.

[0037] In some embodiments, the reporter gene is modified with a luminescent substance, an electrochemical signaling molecule, a Raman signaling molecule, or an electrochemiluminescent signaling molecule;

[0038] In some embodiments, a linker sequence is further included, which is used to connect with the linker regions of probe 1, probe 3, and probe 4 to form a ring. Optionally, the number of bases in which the linker sequence hybridizes complementaryly with linker region I and linker region II is 2-40 nt, more preferably 2-20 nt, and even more preferably 6-8 nt. The number of bases in which the linker sequence does not hybridize complementaryly with linker region I and linker region II and is located in the middle region of the linker sequence is ≥1 nt and ≤20 nt. More preferably, the linker sequences corresponding to probe 1, probe 3, and probe 4 are as shown in SEQ ID NO. 9-11, respectively.

[0039] In some embodiments, the ligase includes T4 DNA ligase or E. coli ligase;

[0040] In some embodiments, the RNA polymerase includes T7 RNA polymerase.

[0041] The embodiments disclosed in this invention relate to the use of the dumbbell-shaped nucleic acid probe or the target detection kit based on the dumbbell-shaped nucleic acid probe in nucleic acid detection or the preparation of nucleic acid detection products.

[0042] In some embodiments, the nucleic acid detection includes the detection of viral genes, miRNAs, bacterial nucleic acids, and tumor-related markers, such as the viral gene HIV, miRNA-21, Staphylococcus aureus, and the tumor-related marker ctDNA.

[0043] The embodiments disclosed in this invention relate to a target detection method based on a dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes, comprising:

[0044] (1) Using the dumbbell-shaped nucleic acid probe or the target detection kit based on the dumbbell-shaped nucleic acid probe, add the sample to be tested and wait for the dumbbell-shaped nucleic acid probe to form a circular template for rolling circle transcription.

[0045] (2) Using the circular template for rolling circle transcription obtained in step (1), perform rolling circle amplification transcription;

[0046] (3) Use the rolling circle amplification transcription product obtained in step (2) to detect the CRISPR-Cas system.

[0047] In some implementations, when the dumbbell-shaped nucleic acid probe is selected from probe 1, probe 3, or probe 4:

[0048] In step (1): the sample to be tested and the probe are added to the buffer solution for the first reaction; after the reaction, the ligation sequence and ligase are added to the obtained reaction solution for the second reaction; optionally, the molar ratio of the probe to the ligation sequence is 4-8:1-2; optionally, the conditions for the first reaction are reaction at 10-70℃ for >2 min, preferably reaction at 37.1-37.5℃ for 1-1.5 h; optionally, the conditions for the second reaction are reaction at 10-70℃ for >2 min, preferably incubation at 37.1-37.5℃ for 0.5-1 h.

[0049] Alternatively, when selecting probe 2 for the dumbbell-shaped nucleic acid probe:

[0050] In step (1): the sample to be tested, the probe and the ligase are added to the buffer solution for reaction; optionally, the reaction conditions are 10-70℃ for >2 min, preferably 37.1-37.5℃ for 1.5-2 h.

[0051] As a preferred implementation method, when selecting probe 1, probe 3, or probe 4 for the dumbbell-shaped nucleic acid probe:

[0052] In step (1): the sample to be tested (concentration of 2 μM, volume range of 1 to 2 μL) and the probe (concentration of 2 μM, volume range of 2 to 4 μL) are added to 1×T4 buffer (volume range of 0.5 to 1 μL) and reacted at 37.0 to 37.5 °C for 1 to 1.5 h;

[0053] Add the reaction mixture containing the ligation sequence (1 μM, 1–2 μL), 1×T4 reaction buffer (0.5–1 μL), and T4 DNA ligase (40–60 U / μL, 0.25–0.5 μL), and incubate at 37.0–37.5 °C for 0.5–1 h.

[0054] Alternatively, when selecting probe 2 for the dumbbell-shaped nucleic acid probe:

[0055] In step (1): the sample to be tested (concentration of 2 μM, volume range of 1-2 μL), probe 2 (concentration of 2 μM, volume range of 2-4 μL), and T4 DNA ligase (enzyme activity of 40-60 U / μL, volume range of 0.25-0.5 μL) are added to 1×T4 buffer (volume range of 0.5-1 μL) and reacted at 37.0-37.5℃ for 1-2 h.

[0056] In some embodiments, in step (2): a reaction mixture containing T7 RNA polymerase, NTP and buffer is added to the reaction product of step (1), and then the reaction is carried out; optionally, the reaction conditions are a reaction at 10-70°C for >2 min, preferably an incubation at 37.0-37.5°C for 1-1.5 h.

[0057] In a preferred embodiment, in step (2): a reaction mixture containing T7 RNA polymerase (15-25 U / μL, volume range 0.5-1 μL), NTP (100 nM for each base, volume range 1-2 μL) and 1×RNAPol buffer (volume range 0.5-1 μL) is added to the reaction product of step (1), and incubated at 37.0-37.5°C for 1-1.5 h.

[0058] In some embodiments, in step (3): an activation sequence, a reporter gene, a Cas protein, and a buffer solution are added to the reaction product of step (2), and the reaction is carried out; optionally, the molar ratio of the activation sequence, the reporter gene, and the Cas protein is in the range of 1000-2000: 200-400: 50-100; optionally, the reaction conditions are a reaction at 10-70°C for >2 min, preferably an incubation at 37.0-37.5°C for 25-30 min.

[0059] In a preferred embodiment, in step (3): an activation sequence (500 nM, volume range 2-4 μL), a reporter gene (200 nM, volume range 1-2 μL), Cas12a (100 nM, volume range 0.5-1 μL), and 1×NEBuffer 2.1 (volume range 1-2 μL) are added to the reaction product of step (2), and the mixture is incubated at 37.0-37.5 °C for 25-30 min.

[0060] The technical solution of this invention has the following advantages:

[0061] 1. The multifunctional dumbbell-shaped nucleic acid probe provided by the present invention includes the following functional regions: a promoter region for binding to RNA polymerase; a target recognition region for recognizing and binding to a target sequence and opening the dumbbell-shaped nucleic acid probe; the dumbbell-shaped nucleic acid probe can bind to rolling loop transcription to perform large-scale transcription of a specific sequence, thereby amplifying the specific sequence, and finally amplifying the signal of the target sequence during detection (e.g., using a CRISPR-Cas detection system). It has the advantages of simple reaction system, low background signal, and high detection specificity. Moreover, the dumbbell-shaped nucleic acid probe can be designed according to different target sequences, enabling controllable detection of different targets.

[0062] 2. The dumbbell-shaped nucleic acid probe provided by the present invention includes at least one of the following probes: probe 1, wherein probe 1 comprises, from the 5' end to the 3' end, a linker region I, a T7 promoter region, a target recognition region, a crRNA direct repeat sequence transcription complementary region, and a linker region II; the linker region I and the linker region II respectively partially complementarily hybridize with the target recognition region to form a stem region, and the T7 promoter region and the crRNA direct repeat sequence transcription complementary region respectively form a circular region;

[0063] Probe 2, from its 5' end to its 3' end, comprises a target recognition region I, a T7 promoter region, an activation sequence complementary hybridization region, a crRNA direct repeat sequence transcription complementary region, and a target recognition region II. The target recognition region I partially hybridizes with the T7 promoter region to form a stem region, the activation sequence complementary hybridization region partially hybridizes with the target recognition region II to form a stem region, and a portion of the T7 promoter region and the crRNA direct repeat sequence transcription complementary region each form a circular region. Optionally, the length of the activation sequence complementary hybridization region is ≥21 nt and ≤25 nt.

[0064] Probe 3, from its 5' end to its 3' end, comprises a linker region I, a target recognition region, a T7 promoter region, a crRNA direct repeat sequence transcription complementation region, and a linker region II. The linker region I and the target recognition region partially complement each other to form a stem region, the linker region II and the T7 promoter region partially complement each other to form a stem region, the target recognition region forms a stem-loop region, and the crRNA direct repeat sequence transcription complementation region forms a loop region.

[0065] Probe 4, from its 5' end to its 3' end, includes a linker region I, a crRNA direct repeat sequence transcription complementary region, a target recognition region, a T7 promoter region, and a linker region II; the linker region I and the linker region II respectively partially complement each other with the target recognition region to form a stem region, and the crRNA direct repeat sequence transcription complementary region and the T7 promoter region respectively form a loop region;

[0066] The aforementioned dumbbell-shaped nucleic acid probe, combined with rolling circle transcription (RCT), generates a repetitive crRNA sequence. The addition of an activation sequence allows for simultaneous transcription and activation of the Cas protein, significantly reducing background signal and enhancing detection specificity, accuracy, and sensitivity. This addresses the high background signal issue of the previously pre-assembled Cas-crRNA complex, significantly improving detection accuracy, sensitivity, and specificity. Furthermore, since the added activation sequence is a single-stranded sequence serving as the target for crRNA recognition, activation of the single-stranded sequence does not require a PAM site, thus easing the stringent requirements for PAM sites in the target dsDNA sequence and greatly expanding the CRISPR-Cas-based nucleic acid sensing toolkit.

[0067] Furthermore, the T7 promoter region and crRNA region of the aforementioned dumbbell-shaped nucleic acid probe are fixed, while the other regions are designed according to the target sequence. Therefore, the dumbbell-shaped nucleic acid probe can controllably detect different targets. Attached Figure Description

[0068] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0069] Figure 1 This is a simulation diagram of the secondary structure prediction of probe 1 in Embodiment 1 of the present invention;

[0070] Figure 2 This is a simulation diagram of the secondary structure prediction of probe 2 in Embodiment 1 of the present invention;

[0071] Figure 3 This is a simulation diagram of the secondary structure prediction of probe 3 in Embodiment 1 of the present invention;

[0072] Figure 4 This is a simulation diagram of the secondary structure prediction of probe 5 in Embodiment 1 of the present invention;

[0073] Figure 5 This is a schematic diagram illustrating the principle of dumbbell-shaped nucleic acid probe detection in Embodiment 1 of the present invention;

[0074] Figure 6 This is the result of PAGE gel electrophoresis detection in Example 3 of the present invention;

[0075] Figure 7 This refers to the fluorescence detection experiment results in Example 3 of the present invention;

[0076] Figure 8 This is the result of PAGE gel electrophoresis detection in Example 6 of the present invention;

[0077] Figure 9 This refers to the fluorescence detection experiment results in Example 6 of the present invention;

[0078] Figure 10 This is the result of PAGE gel electrophoresis detection in Example 9 of the present invention;

[0079] Figure 11 This refers to the detection results of target fluorescence at different concentrations using probe 1 in Example 14 of this invention;

[0080] Figure 12 This is the fluorescence detection result of probe 2 detecting targets at different concentrations in Example 15 of the present invention;

[0081] Figure 13 This is the verification result of the specificity of probe 1 in detecting different targets in Embodiment 17 of the present invention;

[0082] Figure 14 This is the verification result of the specificity of probe 2 in detecting different targets in Embodiment 17 of the present invention;

[0083] Figure 15 This is the stability verification result of the probe 1 detection system in Embodiment 18 of the present invention;

[0084] Figure 16 This is the stability verification result of the probe 2 detection system in Embodiment 18 of the present invention. Detailed Implementation

[0085] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0086] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0087] 1×T4 buffer was purchased from NEB Corporation, USA;

[0088] T4 DNA ligase was purchased from NEB Corporation, USA.

[0089] T7 RNA polymerase was purchased from NEB Corporation, USA.

[0090] 1×RNAPol buffer was purchased from NEB Corporation, USA;

[0091] 1×NEBuffer 2.1 was purchased from NEB Corporation, USA;

[0092] In the following examples, the RRI is an RNase inhibitor.

[0093] The nucleic acid synthesis involved in the following examples, such as the synthesis of probes 1-6, was performed by Shanghai Sangon Biotech Co., Ltd. After probes 1-6 were synthesized into linear sequences, they were incubated in 1×T4 buffer (purchased from NEB Corporation, USA) at 95°C for 5 min. The mixture was then slowly cooled to 25°C to allow it to perfectly fold into a dumbbell-shaped structure. The resulting dumbbell-shaped substrate probes were stored at -20°C for further use.

[0094] The nucleic acid sequences involved in the following examples are shown in Table 1 below:

[0095] Table 1. Nucleic Acid Sequences

[0096]

[0097]

[0098] Note: In the table above, "" represents the target identification area, and "======" represents the T7 startup area. "Represents the transcribed complementary region of the crRNA direct repeat sequence," “ represents connection area I”, and “-------------” represents connection area II.

[0099] Example 1: Design of dumbbell-shaped nucleic acid probes

[0100] The dumbbell-shaped nucleic acid probe provided in this embodiment includes the following functional areas:

[0101] The promoter region is used to bind to RNA polymerase.

[0102] The target recognition region is used to identify and bind to the target sequence and open the dumbbell-shaped nucleic acid probe.

[0103] Connection area, used to connect to form a ring-shaped template.

[0104] In the aforementioned dumbbell-shaped nucleic acid probes: a target recognition region is used to recognize and bind to the target sequence and open the dumbbell-shaped nucleic acid probe. After the dumbbell-shaped nucleic acid probe is opened, the linker region can connect to form a circular template. In some embodiments, the target recognition region and the linker region can be the same sequence. When the target sequence is recognized and bound to the probe, it can simultaneously act as a linker sequence, connecting the dumbbell-shaped nucleic acid probe to form a circular template, as in probe 2. In some embodiments, the target recognition region and the linker region are not the same sequence; a linker sequence in the reaction system can bind to the linker region on the probe to form a circular template, as in probes 1, 3, 4, 5, and 6. When the dumbbell-shaped nucleic acid probe is opened and a circular template is formed, the RNA polymerase bound to the promoter region can initiate rolling loop transcription along its polymerization direction, generating a large amount of the target sequence, amplifying the target sequence, and ultimately amplifying the target sequence signal during detection.

[0105] As an alternative implementation, the promoter region may be selected from promoter sequences commonly used in the art that can bind to RNA polymerase, including but not limited to the T7 promoter region.

[0106] As an alternative implementation, the promoter region is located upstream of the polymerization direction of RNA polymerase in the dumbbell-shaped nucleic acid probe, which ensures that the RNA polymerase bound to the promoter region can transcribe the downstream target sequence along its polymerization direction.

[0107] In a preferred embodiment, when the promoter region is a T7 promoter region, its position is upstream of the T7 RNA polymerase polymerization direction 5'→3', close to the 5' end of the dumbbell-shaped nucleic acid probe.

[0108] As an alternative implementation, the linker region is located at the 5' and 3' ends of the dumbbell-shaped nucleic acid probe, which ensures that the linker sequence is complementary to the sequence located at the 5' and 3' ends of the dumbbell-shaped nucleic acid probe to form a circular template.

[0109] As an optional implementation, the functional region further includes a crRNA direct repeat sequence transcription complementation region for rolling loop transcription to generate crRNA direct repeat sequences. In the long single-stranded RNA generated after rolling loop transcription, the crRNA direct repeat sequence and its upstream or downstream sequence (as a spacer sequence) form crRNA. For example, in probe 1, the target recognition region serves as the crRNA spacer sequence, and the crRNA direct repeat sequence transcription complementation region serves as the crRNA direct repeat sequence. The transcripts of the target recognition region and the transcripts of the crRNA direct repeat sequence transcription complementation region in the long single-stranded RNA formed after transcription form complete crRNA. An activation sequence is added, which is recognized and complemented by the spacer sequence on the crRNA, activating the CRISPR-Cas reaction. This achieves simultaneous transcription and activation of the Cas protein, significantly reducing background signal and improving detection specificity, accuracy, and sensitivity. In probe 2, the activation sequence complementation hybridization region serves as the crRNA spacer sequence. In probe 3, the T7 promoter region serves as the crRNA spacer sequence. In probe 4, the target recognition region serves as the crRNA spacer sequence.

[0110] As an alternative implementation, the functional regions on the single-stranded nucleotide sequence of the dumbbell-shaped nucleic acid probe are arranged sequentially along the direction 5'→3', and self-assembled to form a dumbbell-shaped nucleic acid probe by the principle of complementary base pairing. When the promoter and target sequence are selected, the sequences of the promoter region and the target recognition region are determined, while the linker region only needs to be designed to be complementary to the promoter region and / or the target recognition region, so that the single-stranded nucleic acid sequence can self-assemble to form a dumbbell-shaped nucleic acid probe.

[0111] In a preferred embodiment, the dumbbell-shaped nucleic acid probe is a DNA sequence.

[0112] In a preferred embodiment, the 5' end of the dumbbell-shaped nucleic acid probe is phosphorylated, and after phosphorylation, it can be linked to the 3' hydroxyl group to form a ring under the action of T4 ligase.

[0113] As an alternative implementation, the dumbbell-shaped nucleic acid probe has the following properties: ΔG < 0 (free energy); Tm: 45-65℃; and / or, GC content: 30-40%. Under these conditions, it can be ensured that the single-stranded nucleotide sequence of the designed dumbbell-shaped nucleic acid probe self-assembles into a dumbbell-shaped nucleic acid probe through the base complementary pairing principle. Therefore, different dumbbell-shaped nucleic acid probes can be designed for different target sequences based on the above design principles. For example, for detecting the targets miR-21 and Staphylococcus aureus, the single-stranded nucleotide sequences of the designed dumbbell-shaped nucleic acid probes are shown as probes 5 and 6 in Table 1, respectively, as well as probes 1-4 designed for HIV viral gene DNA. The secondary structure prediction simulation diagrams of the above probes are shown in [Table 1]. Figure 1-4 .

[0114] As an alternative implementation method, dumbbell-shaped nucleic acid probes were designed targeting HIV viral gene DNA, as shown in Table 1. The specific design and detection principles are as follows:

[0115] The simulation diagrams of the secondary structure predictions for probes 1-6 above are shown below. Figure 1-4 .

[0116] The design and detection principles of the dumbbell-shaped nucleic acid probes described above are as follows: Figure 5 , as follows:

[0117] Design principle of probe 1:

[0118] The probe 1, from its 5' end to its 3' end, sequentially includes a linker region I, a T7 promoter region, a target recognition region, a crRNA direct repeat sequence transcription complementation region, and a linker region II. Linker regions I and II partially hybridize with the target recognition region to form stem regions, while the T7 promoter region and the crRNA direct repeat sequence transcription complementation region form circular regions. The 5' end of the probe is phosphorylated, allowing it to loop with the 3' hydroxyl group under the action of T4 ligase. Wherein:

[0119] The sequences of the T7 promoter region and the transcribed complementary region of the crRNA direct repeat sequence are fixed; the target recognition region and the linker region are variable. The target recognition region and the linker region are designed based on the target sequence. In this embodiment, they are designed for HIV-DNA, but are not limited to the aforementioned target gene and can be replaced with other target genes. Therefore, this probe can be designed as a universal detection probe with a wide range of applications. The T7 promoter region and the transcribed complementary region of the crRNA direct repeat sequence are designed as two loop regions of a dumbbell, with the T7 promoter region closer to the 5' end and the crRNA direct repeat sequence transcribed complementary region closer to the 3' end. Because T7 RNA polymerase polymerizes in the 5'-3' direction, this design theoretically ensures that no high background signal is generated; that is, without a target, T7 RNA polymerase will only polymerize to the 5' end and then stop polymerizing, without producing crRNA.

[0120] Detection principle using probe 1:

[0121] When the target gene (HIV-DNA) is present, it recognizes the target recognition region of probe 1 and binds to probe 1 through complementary base pairing, causing the complementary sequence of the original stem region of probe 1 to break, releasing linker region I and linker region II. Then linker region I and linker region II bind to the added linker sequence 1, and are connected into a circular shape under the action of T4 DNA ligase, which is used as the rolling circle template for subsequent RCT. T7 RNA polymerase and NTP are then added to initiate rolling circle transcription (RCT) for signal amplification, transcribing a long single-stranded RNA containing multiple repeating crRNA sequences (there may be multiple crRNA sequences on a single long single strand, but multiple discontinuous long single strands may be generated during transcription). The crRNA sequence consists of a target recognition region and a complementary sequence to the transcribed complementary region of the crRNA direct repeat sequence. The complementary sequence of the target recognition region serves as the crRNA spacer sequence, and the complementary sequence of the transcribed complementary region of the crRNA direct repeat sequence serves as the crRNA direct repeat sequence. Under the action of the activation sequence and Cas12a enzyme, activation sequence 1 binds complementary to the crRNA spacer sequence, activating the non-specific cleavage activity of Cas12a enzyme, cleaving the reporter gene, and detecting fluorescence intensity. If HIV-DNA is absent, probe 1 cannot be opened, forming a circular shape with the linker sequence, thus preventing RCT, and without crRNA production, no fluorescence signal can be detected.

[0122] Similarly, the target recognition region described above is designed based on the target sequence to be tested. In this embodiment, it is designed for HIV-DNA, but it is not limited to the target gene mentioned above. It can also be replaced with other target genes. Therefore, this probe can be designed as a universal detection probe with a wide range of applications. For example, probe 5 is designed based on the target miRNA-21, and probe 6 is designed based on S. aureus mecAgene, based on the above design and detection principles.

[0123] Design principle of probe 2:

[0124] The probe 2, from its 5' end to its 3' end, sequentially includes a target recognition region I, a T7 promoter region, an activation sequence complementary hybridization region, a crRNA direct repeat sequence transcription complementary region, and a target recognition region II. The target recognition region I partially hybridizes with the T7 promoter region to form a stem region; the activation sequence complementary hybridization region and the target recognition region II partially hybridize to form a stem region; and a portion of the T7 promoter region and the crRNA direct repeat sequence transcription complementary region each form a circular region. Wherein:

[0125] The T7 promoter region is placed in the stem-loop region of the dumbbell structure. Target recognition regions I and II are located in the open arms at both ends of the dumbbell structure. Target recognition region I undergoes base complementation hybridization with a portion of the T7 promoter region to form the stem region of the dumbbell structure. Target recognition region I also undergoes partial complementary hybridization hybridization with the activation sequence to form the stem region of the dumbbell structure. The remaining region of the T7 promoter region serves as the 5' loop region of the dumbbell structure, and the transcribed complementary region of the crRNA direct repeat sequence serves as the 3' loop region of the dumbbell structure. The main feature is that target recognition regions I and II have dual functions: they act as both the activation region triggering the RCT and the linker region connecting the dumbbell structure to form a loop. This reduces target recognition time in experimental procedures, as the target is both the linker sequence and the driving sequence for opening the probe, saving time. After adding the target, there is no need to add the linker sequence; simply add T4 DNA ligase to form a loop, simplifying the experimental procedure.

[0126] Similarly, the target recognition region described above is designed based on the target sequence to be tested. In this embodiment, it is designed for HIV-DNA, but it is not limited to the target gene mentioned above. It can also be replaced with other target genes. Therefore, this probe can be designed as a universal detection probe with a wide range of applications.

[0127] Utilizing the RCT detection principle of probe 2:

[0128] When probe 2 recognizes the target HIV-DNA, the dumbbell structure opens under the base complementarity force, and is further ligated into a rolling circle template by T4 DNA ligase for subsequent RCT. T7 RNA polymerase and NTP are then added to initiate the rolling circle amplification transcription reaction (RCT) for signal amplification, subsequently transcribing a long single-stranded RNA containing multiple repeating crRNA sequences (possibly multiple crRNA sequences on a single long single strand, but multiple discontinuous long single strands may be generated during transcription). The crRNA sequence consists of complementary sequences from the activation sequence complementary hybridization region and the crRNA direct repeat transcribed complementary region. The complementary sequence of the activation sequence complementary hybridization region serves as the crRNA spacer sequence, and the complementary sequence of the crRNA direct repeat transcribed complementary region serves as the crRNA direct repeat sequence. Through activation sequence 2 and Cas12a enzyme, activation sequence 2 binds to the crRNA spacer sequence, further activating the non-specific cleavage activity of Cas12a. The reporter gene in the system is cleaved, subsequently emitting fluorescence. The detection limit and linear range are calculated based on the fluorescence intensity measurement to evaluate the detection sensitivity and specificity of this biosensor. If there is no target in the system, the dumbbell structure will remain stable. When T7 RNA polymerase is added, crRNA will not be produced due to the direction of polymerization, so there is no background signal interference, resulting in better and more reliable detection performance.

[0129] Design principle of probe 3:

[0130] The probe 3, from its 5' end to its 3' end, sequentially includes a linker region I, a target recognition region, a T7 promoter region, a crRNA direct repeat sequence transcription complementation region, and a linker region II. Linker region I and the target recognition region partially hybridize to form a stem region; linker region II and the T7 promoter region partially hybridize to form a stem region; the target recognition region forms a stem-loop region; and the crRNA direct repeat sequence transcription complementation regions each form a loop region. The 5' end of the probe is phosphorylated, allowing it to loop with the 3' hydroxyl group under the action of T4 ligase.

[0131] The T7 promoter region and the transcribed complementary region of the crRNA direct repeat sequence are adjacent and placed in the stem region and 3' loop region of the dumbbell structure, respectively. The target recognition region is located in the 5' stem-loop region of the dumbbell structure. The stem-loop region is formed by the 5' end sequence of the target recognition region forming a loop, and the 3' end sequence forming a partial stem region (complementary hybridization with linker region I). Linker regions I and II are placed in the two open arm regions (5' end and 3' end) of the dumbbell structure and form stem regions by base complementarity hybridization with the target recognition region and the T7 promoter region, respectively.

[0132] Similarly, the target recognition region described above is designed based on the target sequence to be tested. In this embodiment, it is designed for HIV-DNA, but it is not limited to the target gene mentioned above. It can also be replaced with other target genes. Therefore, this probe can be designed as a universal detection probe with a wide range of applications.

[0133] Detection principle using probe 3:

[0134] When probe 3 recognizes the target HIV-DNA, the target HIV-DNA binds to the base-complementary region (target recognition region) located at the 5' end. Under the base complementarity force, the stem-loop complementary pairing structure at the 5' end of the dumbbell structure is opened, and it is further ligated into a rolling circle template by ligation sequence 2 and T4 DNA ligase for subsequent RCT. Then, T7 is added. RNA polymerase and NTP initiate rolling circle amplification transcription (RCT) to amplify the signal, subsequently transcribing a long single-stranded RNA containing multiple repeating crRNA sequences (possibly multiple crRNA sequences on a single long single strand, but multiple discontinuous long single strands may be generated during transcription). The crRNA sequence is composed of the T7 promoter and the complementary sequence of the transcribed complementary region of the crRNA direct repeat sequence. The complementary sequence of the T7 promoter serves as the crRNA spacer, and the complementary sequence of the transcribed complementary region of the crRNA direct repeat sequence serves as the crRNA direct repeat sequence. Through activation sequence 3 and Cas12a enzyme, activation sequence 3 binds to the crRNA spacer sequence, further activating the non-specific cleavage activity of Cas12a. The reporter probe in the system is cleaved, subsequently emitting fluorescence. The detection limit and linear range are calculated based on the fluorescence intensity measurement to evaluate the detection sensitivity and specificity of this biosensor. If no target is present in the system, the dumbbell-shaped probe remains stable. When T7 RNA polymerase is added, crRNA will not be generated due to the polymerization direction, thus eliminating background signal interference and resulting in superior and reliable detection performance.

[0135] Design principle of probe 4:

[0136] Probe 4, from the 5' end to the 3' end, includes a linker region I, a crRNA direct repeat sequence transcription complementary region, a target recognition region, a T7 promoter region, and a linker region II; the linker region I and the linker region II respectively partially complement each other with the target recognition region to form a stem region, and the crRNA direct repeat sequence transcription complementary region and the T7 promoter region respectively form a loop region.

[0137] The design principle of probe 4 is similar to that of probe 1, except that the T7 promoter region is closer to the 3' end and the transcribed complementary region of the crRNA direct repeat sequence is closer to the 5' end.

[0138] Similarly, the detection principle of probe 4 is similar to that of probe 1. The difference is that the T7 promoter region and the transcribed complementary region of the crRNA direct repeat sequence are designed as two loop regions of a dumbbell. The T7 promoter region is at the 3' end, and the transcribed complementary region of the crRNA direct repeat sequence is closer to the 5' end. Since T7 RNA polymerase polymerizes in the 5'-3' direction, this design may easily generate background signals. That is, when there is no target, T7 RNA polymerase may produce crRNA when it polymerizes to the 5' end.

[0139] Example 2: Target Detection Kit Based on Dumbbell-Shaped Nucleic Acid Probes

[0140] This embodiment provides a target detection kit based on dumbbell-shaped nucleic acid probes, including the following individually packaged components:

[0141] Any one of probes 1 to 6 in Example 1;

[0142] T4 DNA ligase;

[0143] T7 RNA polymerase;

[0144] Cas12a protein;

[0145] The activation sequences, the nucleotide sequences of activation sequences 1 to 6 corresponding to probes 1 to 6 are shown in SEQ ID NO.5 to 8, SEQ ID NO.19 and SEQ ID NO.23 respectively, as shown in Table 1;

[0146] Reporter gene; the sequence of the reporter gene is: 5'-FAM-TTATT-BHQ1-3', where FAM is a fluorescent group and BHQ1 is a quenching group; the two ends of the reporter gene are modified with fluorescent groups FAM (emission wavelength 518nm);

[0147] 1×T4 reaction buffer;

[0148] 1×RNAPol buffer;

[0149] NTP.

[0150] When the probe is probe 1, 3, 4, 5 or 6, it also includes independently packaged connection sequences 1 to 5. The connection sequences are as follows: the connection sequences corresponding to probe 1, probe 3, probe 4, 5 or 6 are shown in SEQ ID NO. 9 to 11, SEQ ID NO. 18 and SEQ ID NO. 22 respectively, see Table 1.

[0151] Example 3: Detecting the target using probe 1

[0152] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0153] (1) Add the sample containing target HIV-DNA (2μM, 1μL), probe 1 (2μM, 2μL) and 1×T4 buffer (0.5μL) to the PCR tube and react at 37℃ for 1 hour to open the dumbbell-shaped probe 1.

[0154] (2) Add the ligation sequence (concentration of 1 μM, volume of 1 μL), T4 DNA ligase (enzyme activity of 40 U / μL, volume of 0.5 μL) and 1×T4 reaction buffer (volume of 0.5 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37°C for 1 hour to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of dumbbell-shaped probe 1. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0155] (3) Add T7 RNA polymerase (20 U / μL, volume 0.5 μL), NTP (100 nM for each base, volume 2 μL) and 1×RNAPol buffer (volume 0.5 μL) to the reaction product of step (2), and incubate at 37°C for 1 hour to complete RCT transcription and achieve first-level signal amplification.

[0156] (4) Add activation sequence 1 (500 nM, 3 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL) and 1×NEBuffer 2.1 (1 μL) to the reaction product of step (3), and incubate at 37°C for 30 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0157] PAGE gel electrophoresis assay:

[0158] The reaction system in each of the following lanes was carried out according to steps (1) to (3) above. The amount of each reagent in the reaction system remained unchanged, and any missing reagents were replaced with an equal volume of sterile, enzyme-free water. The obtained products were subjected to PAGE gel electrophoresis, and the electrophoresis results are shown in [Figure 1]. Figure 5 :

[0159] Lane 0 (far right): 25–500bp DNA ladder marker;

[0160] lane 1: probe 1;

[0161] Lane 2: HIV-DNA;

[0162] lane 3: Connecting sequence;

[0163] Lane 4: Probe 1 + HIV-DNA + T4 buffer;

[0164] Lane 5: Probe 1 + Ligation Sequence + T4 DNA Ligase + T4 Buffer;

[0165] Lane 6: Probe 1 + HIV-DNA + Ligation Sequence + T4 DNA Ligase + T4 Buffer;

[0166] Lane 7: Probe 1 + Ligation Sequence + T4 DNA Ligase + T4 Buffer + T7 RNA Polymerase + rNTPs + RRI.

[0167] Lane 8: Probe 1 + HIV-DNA + Ligation Sequence + T4 DNA Ligase + T4 Buffer + T7 RNA Polymerase + rNTPs + RRI;

[0168] Lane 9: Probe 1 + HIV-DNA + T4 DNA ligase + T4 buffer + T7 RNA polymerase + rNTPs + RRI.

[0169] See the results Figure 6 In the diagram, lane 1 corresponds to the band of probe 1; lane 4 corresponds to the probe 1 + HIV-DNA hybridization complex; in lane 5, due to the lack of HIV-DNA in the reaction system, a circular template for RCT could not be formed; lane 6 corresponds to the circular template used for RCT; in lane 7, due to the lack of HIV-DNA in the reaction system, the RCT reaction could not be performed, and no large long single-stranded RNA sequence was generated; lane 8 has a large long single-stranded RNA sequence at the top; lane 9, due to the lack of a linker sequence, could not perform the RCT reaction, and no large long single-stranded RNA sequence was generated. The above verification shows that probe 1 in this embodiment can only initiate the RCT reaction in the presence of the target sequence and the linker sequence. Without initiation, the lack of a large long single-stranded RNA sequence containing crRNA indicates a low background signal, which can improve the accuracy, specificity, and sensitivity of the detection.

[0170] Fluorescence detection experiment verification:

[0171] The test samples containing target HIV-DNA (2 μM, 1 μL) and the test samples without target (replaced by an equal volume of sterile enzyme-free water) were processed according to steps (1) to (4) above. The results are as follows: Figure 7 As shown, fluorescence signals can be detected when there is a target, and no fluorescence signals are generated when there is no target.

[0172] Example 4: Detecting the target using probe 1

[0173] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0174] (1) Add the sample containing target HIV-DNA (2 μL), probe 1 (2 μM, 4 μL) and 1×T4 buffer (1 μL) to the PCR tube and react at 37.5℃ for 1.5 hours to open the dumbbell-shaped probe 1.

[0175] (2) Add the ligation sequence 1 (concentration of 1 μM, volume of 2 μL), T4 DNA ligase (enzyme activity of 15 U) and 1×T4 reaction buffer (volume of 1 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37.5℃ for 1 hour to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of dumbbell-shaped probe 1. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0176] (3) Add T7 RNA polymerase (25 U / μL, volume 1 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 1 μL) to the reaction product of step (2), and incubate at 37.5℃ for 1.5 hours to complete RCT transcription and achieve first-level signal amplification.

[0177] (4) Add activation sequence 1 (500 nM, 2 μL), reporter gene (200 nM, 2 μL), Cas12a (100 nM, 1 μL) and 1×NEBuffer 2.1 (2 μL) to the reaction product of step (3), and incubate at 37.5 °C for 25 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0178] Example 5: Detecting the target using probe 1

[0179] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0180] (1) Add the sample containing target HIV-DNA (1.5 μL), probe 1 (2 μM, 3 μL) and 1×T4 buffer (0.8 μL) to the PCR tube and react at 37.3℃ for 1.2 hours to open the dumbbell-shaped probe 1;

[0181] (2) Add ligation sequence 1 (concentration of 1 μM, volume of 1.5 μL), T4 DNA ligase (enzyme activity of 20 U) and 1×T4 reaction buffer (volume of 0.8 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37.3℃ for 0.8 hours to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of dumbbell-shaped probe 1. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0182] (3) Add T7 RNA polymerase (20 U / μL, volume 0.8 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 0.8 μL) to the reaction product of step (2), and incubate at 37.3℃ for 1.2 hours to complete RCT transcription and achieve first-level signal amplification;

[0183] (4) Add activation sequence 1 (500 nM, 4 μL), reporter gene (200 nM, 1.5 μL), Cas12a (100 nM, 0.8 μL) and 1×NEBuffer 2.1 (1.5 μL) to the reaction product of step (3), and incubate at 37.3 °C for 28 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0184] Example 6: Detecting the target using probe 2

[0185] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0186] (1) Add the sample containing target HIV-DNA (2μM, 2μL), probe 2 (2μM, 2μL), T4 DNA ligase (60U / μL, 0.5μL) and 1×T4 buffer (0.5μL) to the PCR tube and react at 37℃ for 1 hour. Under the action of T4 DNA ligase, the dumbbell structure formed after ligation forms the circular template of RCT.

[0187] (2) Add T7 RNA polymerase (20 U / μL, volume 0.5 μL), NTP (100 nM for each base, volume 2 μL) and 1×RNAPol buffer (volume 0.5 μL) to the reaction product of step (1), and incubate at 37°C for 1 hour to complete RCT transcription and achieve first-level signal amplification.

[0188] (3) Add activation sequence 2 (500 nM, 3 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL) and 1×NEBuffer 2.1 (1 μL) to the reaction product of step (2), and incubate at 37°C for 30 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0189] PAGE gel electrophoresis assay:

[0190] The reaction system in each of the following lanes was carried out according to steps (1) to (3) above. The amount of each reagent in the reaction system remained unchanged, and any missing reagents were replaced with an equal volume of sterile, enzyme-free water. The obtained products were subjected to PAGE gel electrophoresis, and the electrophoresis results are shown in [Figure 1]. Figure 8 :

[0191] Lane 0 (far right): 25–500bp DNA ladder marker;

[0192] lane 1: probe 2;

[0193] Lane 2: HIV-DNA;

[0194] Lane 3: Probe 2 + HIV-DNA + T4 DNA ligase + T4 buffer;

[0195] Lane 4: Probe 2 + T4 DNA ligase + T4 buffer;

[0196] Lane 5: Probe 2 + HIV-DNA + T4 DNA ligase + T4 buffer + T7 RNA polymerase + rNTPs + RRI.

[0197] Lane 6: Probe 2 + T4 DNA ligase + T4 buffer + T7 RNA polymerase + rNTPs + RRI;

[0198] Lane 7: Probe 2 + T4 DNA ligase + T7 RNA polymerase + rNTPs + RRI.

[0199] Lane 8: Probe 2 + HIV-DNA + T7 RNA polymerase + rNTPs + RRI.

[0200] See results Figure 7 In the sample, lane 1 corresponds to the band of probe 2 (probe 2 may form a hairpin secondary structure, which migrates slower in the gel than the linear strand, so the band in lane 1 is slightly higher than 100 nt); lane 3 corresponds to the complex formed by the ligation of the target and probe 2, forming a circular template for RCT; lane 4 lacks a target and only corresponds to probe 2; lane 5 has a long single-stranded RNA sequence at the top that produces a large fragment; lane 6 lacks a target sequence, failed to form a circular template for RCT, failed to perform the RCT reaction, and did not produce a large single-stranded RNA sequence; lane 7 lacks a target sequence, failed to form a circular template for RCT, failed to perform the RCT reaction, and did not produce a large single-stranded RNA sequence; lane 8 lacks a ligase, failed to form a circular template for RCT, failed to perform the RCT reaction, and did not produce a large single-stranded RNA sequence. The above verification shows that probe 2 in this embodiment can only initiate the RCT reaction in the presence of the target sequence and ligase. Without initiation, no large fragments of long single-stranded RNA sequences containing crRNA are generated, indicating a low background signal, which can improve the accuracy, specificity and sensitivity of detection.

[0201] Fluorescence detection experiment verification:

[0202] The test samples containing HIV-DNA (2 μM, 2 μL) and those without the target (replaced by an equal volume of sterile, enzyme-free water) were processed according to steps (1) to (4) above. The results are as follows: Figure 9 As shown, fluorescence signals can be detected when there is a target, and no fluorescence signals are generated when there is no target.

[0203] Example 7: Detecting the target using probe 2

[0204] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0205] (1) Add the sample containing target HIV-DNA (1 μL), probe 2 (2 μM, 4 μL), T4 DNA ligase (10 U) and 1×T4 buffer (1 μL) to the PCR tube and react at 37.5℃ for 2 hours. Under the action of T4 DNA ligase, the dumbbell structure formed after ligation forms the circular template of RCT.

[0206] (2) Add T7 RNA polymerase (25 U / μL, volume 1 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 1 μL) to the reaction product of step (1), and incubate at 37.5℃ for 1.5 hours to complete RCT transcription and achieve first-level signal amplification.

[0207] (3) Add activation sequence 2 (500 nM, 2 μL), reporter gene (200 nM, 2 μL), Cas12a (100 nM, 1 μL) and 1×NEBuffer 2.1 (2 μL) to the reaction product of step (2), and incubate at 37.5 °C for 25 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0208] Example 8: Detecting the target using probe 2

[0209] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0210] (1) Add the sample containing target HIV-DNA (1.5 μL), probe 2 (2 μM, 3 μL), T4 DNA ligase (20 U) and 1×T4 buffer (0.8 μL) to the PCR tube and react at 37.3℃ for 1.5 hours. The dumbbell structure formed by the ligation under the action of T4 DNA ligase forms the circular template of RCT.

[0211] (2) Add T7 RNA polymerase (20 U / μL, volume 0.8 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 0.8 μL) to the reaction product of step (1), and incubate at 37.3℃ for 1.3 hours to complete RCT transcription and achieve first-level signal amplification;

[0212] (3) Add activation sequence 2 (500 nM, 4 μL), reporter gene (200 nM, 1.5 μL), Cas12a (100 nM, 0.8 μL), and 1×NEBuffer 2.1 (1.5 μL) to the reaction product of step (2), and incubate at 37.3 °C for 28 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0213] Example 9: Detecting the target using probe 3

[0214] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0215] (1) Add the sample containing target HIV-DNA (2μM, 2μL), probe 3 (2μM, 2μL) and 1×T4 buffer (0.5μL) to the PCR tube and react at 37℃ for 1 hour to open the dumbbell-shaped probe 3.

[0216] (2) Add the ligation sequence 3 (concentration of 2 μM, volume of 1 μL), T4 DNA ligase (enzyme activity of 40 U / μL, volume of 0.5 μL) and 1×T4 reaction buffer (volume of 0.5 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37°C for 1 hour to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of the dumbbell-shaped probe 3. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0217] (3) Add T7 RNA polymerase (20 U / μL, volume 0.5 μL), NTP (100 nM for each base, volume 2 μL) and 1×RNAPol buffer (volume 0.5 μL) to the reaction product of step (2), and incubate at 37°C for 1 hour to complete RCT transcription and achieve first-level signal amplification.

[0218] (4) Add activation sequence 3 (500 nM, 3 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL) and 1×NEBuffer 2.1 (1 μL) to the reaction product of step (3), and incubate at 37°C for 30 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0219] PAGE gel electrophoresis assay:

[0220] The reaction system in each of the following lanes was carried out according to steps (1) to (3) above. The amount of each reagent in the reaction system remained unchanged, and any missing reagents were replaced with an equal volume of sterile, enzyme-free water. The obtained products were subjected to PAGE gel electrophoresis, and the electrophoresis results are shown in [Figure 1]. Figure 10 :

[0221] Lane 0 (far right): 25–500bp DNA ladder marker;

[0222] lane 1: probe 3;

[0223] Lane 2: HIV-DNA;

[0224] lane 3: Connecting sequence;

[0225] Lane 4: Probe 3 + HIV-DNA + T4 buffer;

[0226] Lane 5: Probe 3 + ligation sequence + T4 DNA ligase + T4 buffer;

[0227] Lane 6: Probe 3 + HIV-DNA + Ligation Sequence + T4 DNA Ligase + T4 Buffer;

[0228] Lane 7: Probe 3 + Ligation Sequence + T4 DNA Ligase + T4 Buffer + T7 RNA Polymerase + rNTPs + RRI.

[0229] Lane 8: Probe 3 + HIV-DNA + T4 DNA ligase + T4 buffer + T7 RNA polymerase + rNTPs + RRI;

[0230] Lane 9: Probe 3 + HIV-DNA + Ligation Sequence + T4 DNA Ligase + T4 Buffer + T7 RNA Polymerase + rNTPs + RRI.

[0231] See results Figure 10In the diagram, lane 1 corresponds to the band of probe 3; lane 4 corresponds to the probe 3 + HIV-DNA hybridization complex; in lane 5, due to the lack of HIV-DNA in the reaction system, a circular template for RCT was not formed; lane 6 corresponds to the circular template used for RCT; in lane 7, due to the lack of HIV-DNA in the reaction system, the RCT reaction failed, and no large long single-stranded RNA sequence was generated; in lane 8, due to the lack of a linker sequence, the RCT reaction failed, and no large long single-stranded RNA sequence was generated; lane 9 has a large long single-stranded RNA sequence at the top. This verifies that probe 3 in this embodiment can only initiate the RCT reaction in the presence of the target sequence and the linker sequence. Without initiation, the lack of a large long single-stranded RNA sequence containing crRNA indicates a low background signal, which can improve the accuracy, specificity, and sensitivity of the detection.

[0232] Fluorescence detection experiment verification:

[0233] The test samples containing HIV-DNA (2 μM, 2 μL) and the test samples without the target (replaced by sterile, enzyme-free water of equal volume) were processed according to the above steps (1) to (4). The results showed that a fluorescent signal could be detected when there was a target, and no fluorescent signal was generated when there was no target.

[0234] Example 10: Detecting a target using probe 3

[0235] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0236] (1) Add the sample containing target HIV-DNA (2 μL), probe 3 (2 μM, 4 μL) and 1×T4 buffer (1 μL) to the PCR tube and react at 37.5℃ for 1.5 hours to open the dumbbell-shaped probe 1.

[0237] (2) Add the ligation sequence 3 (concentration of 1 μM, volume of 2 μL), T4 DNA ligase (enzyme activity of 15 U) and 1×T4 reaction buffer (volume of 1 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37.5℃ for 1 hour to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of dumbbell-shaped probe 1. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0238] (3) Add T7 RNA polymerase (25 U / μL, volume 1 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 1 μL) to the reaction product of step (2), and incubate at 37.5℃ for 1.5 hours to complete RCT transcription and achieve first-level signal amplification.

[0239] (4) Add activation sequence 3 (500 nM, 2 μL), reporter gene (200 nM, 2 μL), Cas12a (100 nM, 1 μL) and 1×NEBuffer 2.1 (2 μL) to the reaction product of step (3), and incubate at 37.5 °C for 25 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0240] Example 11: Detecting a target using probe 3

[0241] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0242] (1) Add the sample containing target HIV-DNA (1.5 μL), probe 3 (2 μM, 3 μL) and 1×T4 buffer (0.8 μL) to the PCR tube and react at 37.3℃ for 1.2 hours to open the dumbbell-shaped probe 1;

[0243] (2) Add ligation sequence 3 (concentration of 1 μM, volume of 1.5 μL), T4 DNA ligase (enzyme activity of 20 U) and 1×T4 reaction buffer (volume of 0.8 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37.3℃ for 0.8 hours to make the ligation sequence complementary to the two open arm regions (5' end and 3' end) of dumbbell probe 1. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0244] (3) Add T7 RNA polymerase (20 U / μL, volume 0.8 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 0.8 μL) to the reaction product of step (2), and incubate at 37.3℃ for 1.2 hours to complete RCT transcription and achieve first-level signal amplification;

[0245] (4) Add activation sequence 3 (500 nM, 4 μL), reporter gene (200 nM, 1.5 μL), Cas12a (100 nM, 0.8 μL) and 1×NEBuffer 2.1 (1.5 μL) to the reaction product of step (3), and incubate at 37.3 °C for 28 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure HIV-DNA.

[0246] Example 12: Detecting the target using probe 5

[0247] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0248] (1) Add the sample containing target miRNA-21 (1 μL), probe 5 (2 μM, 2 μL) and 1×T4 buffer (0.5 μL) to the PCR tube and react at 37°C for 1 hour to open the dumbbell-shaped probe 5.

[0249] (2) Add the ligation sequence 4 (concentration of 1 μM, volume of 1 μL), T4 DNA ligase (enzyme activity of 40 U / μL, volume of 0.5 μL) and 1×T4 reaction buffer (volume of 0.5 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37°C for 1 hour, so that the ligation sequence 4 is complementary to the two open arm regions (5' end and 3' end) of the dumbbell-shaped probe 5. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0250] (3) Add T7 RNA polymerase (20 U / μL, volume 0.5 μL), NTP (100 nM for each base, volume 2 μL) and 1×RNAPol buffer (volume 0.5 μL) to the reaction product of step (2), and incubate at 37°C for 1 hour to complete RCT transcription and achieve first-level signal amplification.

[0251] (4) Add activation sequence 5 (500 nM, 3 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL) and 1×NEBuffer 2.1 (1 μL) to the reaction product of step (3), and incubate at 37°C for 30 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure miRNA-21.

[0252] Example 13: Target detection using probe 6

[0253] This embodiment provides a target detection method based on dumbbell-shaped nucleic acid probes for non-disease diagnosis or treatment purposes, using the kit from Example 2 for detection, and includes the following steps:

[0254] (1) Add the sample containing the target S.aureus mecAgene (1 μL), probe 5 (2 μM, 2 μL) and 1×T4 buffer (0.5 μL) to the PCR tube and react at 37°C for 1 hour to open the dumbbell-shaped probe 5.

[0255] (2) Add the ligation sequence 5 (concentration of 1 μM, volume of 1 μL), T4 DNA ligase (enzyme activity of 20 U) and 1×T4 reaction buffer (volume of 0.5 μL) to the reaction product in step (1) to carry out the T4 DNA ligase-mediated ligation reaction. The reaction is carried out at 37°C for 1 hour, so that the ligation sequence 5 is complementary to the two open arm regions (5' end and 3' end) of the dumbbell-shaped probe 5. Under the action of T4 DNA ligase, the dumbbell structure after ligation forms the circular template of RCT.

[0256] (3) Add T7 RNA polymerase (20 U / μL, volume 0.5 μL), NTP (concentration of 100 nM for each base) and 1×RNAPol buffer (volume 0.5 μL) to the reaction product of step (2), and incubate at 37°C for 1 hour to complete RCT transcription and achieve first-level signal amplification.

[0257] (4) Add activation sequence 6 (500 nM, 3 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL) and 1×NEBuffer 2.1 (1 μL) to the reaction product of step (3), and incubate at 37°C for 30 minutes; use a fluorescence analyzer FL970PLUS (purchased from Tianmei Group) to record the fluorescence emission spectrum from 505 nm to 600 nm when excited at 488 nm, and collect the fluorescence intensity at 520 nm to measure S. aureus mecA gene.

[0258] Experiment 14: Probe 1 detects target concentration gradient

[0259] The difference between this embodiment and Example 3 is that the sample to be tested is replaced with target HIV-DNA and diluted with DEPC H2O to concentrations of 50nM, 25nM, 1nM, 0.5nM, 0.1nM, 50pM, 10pM, 5pM, 1pM, 100fM, 10fM, and 1fM, respectively, to obtain HIV-DNA dilutions of different concentrations.

[0260] Fluorescence detection results as follows Figure 11 As shown, the probe 1 can detect signal values ​​of target HIV-DNA at different concentrations, and can assess HIV infection at different levels, with a detection limit of 1 fM.

[0261] Experiment 15: Probe 2 detects target concentration gradient

[0262] The difference between this embodiment and Example 6 is that the test sample is replaced with target HIV-DNA and diluted with DEPC H2O to concentrations of 50nM, 25nM, 1nM, 0.5nM, 0.1nM, 50pM, 10pM, 5pM, 1pM, 100fM, 10fM, and 1fM, respectively, to obtain HIV-DNA dilutions of different concentrations.

[0263] Fluorescence detection results as follows Figure 12 As shown, the probe 2 can detect signal values ​​of target HIV-DNA at different concentrations, and can assess HIV infection at different levels, with a detection limit of 4.28 fM.

[0264] Experiment 16: Detection of target concentration gradient using probe 3

[0265] The difference between this embodiment and Example 10 is that the test sample is replaced with target HIV-DNA and diluted with DEPC H2O to concentrations of 50nM, 25nM, 1nM, 0.5nM, 0.1nM, 50pM, 10pM, 5pM, 1pM, 100fM, 10fM, and 1fM, respectively, to obtain HIV-DNA dilutions of different concentrations.

[0266] According to the fluorescence detection results, the probe 3 can detect the signal value of target HIV-DNA at different concentrations, which can be used to assess the HIV infection status at different levels, with a minimum detection limit of 10 fM.

[0267] Example 17 Specificity Validation of the Probe Detection System

[0268] To assess specificity, this study used interfering DNA (such as HIV-1-gag-DNA) and three similar HCV-DNAs available in the laboratory as models.

[0269] For probe 1 specificity verification, the method was as described in Example 3, with the test samples replaced with 1 μM of different target nucleic acids. The targets were replaced with HIV-DNA, HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k, respectively. 1×T4 buffer was used as a blank control, and the mixture consisted of HIV-DNA, HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k, with a final concentration of 1 μM for each target nucleic acid.

[0270] Depend on Figure 13 It can be seen that the FAM fluorescence signals in the presence of HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k are the same as those in the blank; in the presence of HIV DNA, the FAM fluorescence signal is significantly enhanced.

[0271] For probe 2 specificity verification, the method was as described in Example 6, with the test samples replaced with 1 μM of different target nucleic acids. The targets were replaced with HIV-DNA, HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k, respectively. 1×T4 buffer was used as a blank control, and the mixture consisted of HIV-DNA, HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k, with a final concentration of 1 μM for each target nucleic acid.

[0272] Depend on Figure 14 It can be seen that the FAM fluorescence signals in the presence of HIV-1-gag-DNA, HCV-DNA, HCV-1b, and HCV-6k are the same as those in the blank; in the presence of HIV DNA, the FAM fluorescence signal is significantly enhanced.

[0273] Similarly, probes 3 and 4 also have specificity.

[0274] Example 18: Stability Verification of the Probe Detection System

[0275] To assess the system stability, the anti-interference performance was studied using T4 buffer and fetal bovine serum samples. The validation process is as follows:

[0276] Two sets of samples were prepared. A 10 μL mixture of 2 μM dumbbell-shaped nucleic acid probe, 1 μM target HIV-DNA, 40 U T4 ligase, 25 U T7 RNA polymerase, and 1 μM of the corresponding ligation sequence was added. One set of samples was treated with 10% fetal bovine serum (FBS), while the other set served as a control without FBS (but with T4 ligase buffer). The volume was brought up to 10 μL with DEPC H2O. The mixture was incubated at 37°C for 3 hours. The control sample did not contain target HIV-DNA. RCT transcription was then completed. Finally, the corresponding activation sequence (1 μM, 1 μL), reporter gene (200 nM, 1 μL), Cas12a (100 nM, 0.5 μL), and 1×NEBuffer were added. 2.1 (volume 1 μL), incubated at 37°C for 30 minutes; using a fluorescence analyzer FL970PLUS (purchased from Tianmei Group), when excited at 488 nm, the fluorescence emission spectrum from 505 to 600 nm was recorded, and the fluorescence intensity at 520 nm was collected.

[0277] For probes 1-2, by Figures 15-16 It can be seen that when HIV DNA is present, a certain FAM signal can be observed even in the above-mentioned unrelated interference mixture, which indicates that the constructed probe and detection system have good specificity and anti-interference ability.

[0278] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A dumbbell-shaped nucleic acid probe, characterized in that: Includes the following functional areas: The promoter region is used for binding of RNA polymerase; The target recognition region is used to recognize and bind to the target sequence and open the dumbbell-shaped nucleic acid probe.

2. The dumbbell-shaped nucleic acid probe according to claim 1, characterized in that: Also includes: The linker region is used for linking to form a circular template.

3. The dumbbell-shaped nucleic acid probe according to claim 1 or 2, characterized in that: The promoter region includes an RNA polymerase promoter region; Optionally, the promoter region is located upstream in the polymerization direction of RNA polymerase in the dumbbell-shaped nucleic acid probe.

4. The dumbbell-shaped nucleic acid probe according to any one of claims 2 to 3, characterized in that: The connecting region is located at the 5' end and the 3' end of the dumbbell-shaped nucleic acid probe.

5. The dumbbell-shaped nucleic acid probe according to any one of claims 1 to 4, characterized in that: The functional area also includes an amplification functional area, which is used to combine with nucleic acid amplification technology to achieve signal generation and amplification.

6. The dumbbell-shaped nucleic acid probe according to any one of claims 1 to 5, characterized in that: The functional regions on the single-stranded nucleotide sequence of the dumbbell-shaped nucleic acid probe are arranged sequentially along the direction 5'→3', and self-assemble to form the dumbbell-shaped nucleic acid probe through the principle of complementary base pairing; or The dumbbell-shaped nucleic acid probe is a DNA sequence; or The 5' end of the dumbbell-shaped nucleic acid probe is phosphorylated.

7. The dumbbell-shaped nucleic acid probe according to any one of claims 1 to 6, characterized in that: In the dumbbell-shaped nucleic acid probe: ΔG<0; Tm: 10-90℃.

8. A target detection kit based on a dumbbell-shaped nucleic acid probe, characterized in that: include: The dumbbell-shaped nucleic acid probe, ligase, RNA polymerase, activation sequence, buffer, NTP and / or reporter gene according to any one of claims 1 to 7.

9. The target detection kit based on dumbbell-shaped nucleic acid probe according to claim 8, characterized in that: It also includes Cas family proteins; The Cas family protein is selected from Cas12a, Cas13a, Cas9 or Cas14; and / or, the activation sequence can be recognized by a sequence upstream of the crRNA direct repeat sequence transcription complementary region, optionally, by a target recognition region, an activation sequence complementary hybridization region or a promoter region; And / or, the reporter gene is modified with a luminescent substance, an electrochemical signal molecule, a Raman signal molecule or an electrochemiluminescent signal molecule; And / or, further comprising a connecting sequence, wherein the connecting sequence is used to complementarily hybridize with the connecting regions of the probe 1, the probe 3, and the probe 4 to form a circular shape; optionally, the number of bases of the connecting sequence complementary hybridizing with the connecting region I and the connecting region II, respectively, is 2-40 nt; and / or, the ligase comprises T4 DNA ligase or E. coli ligase; And / or, the RNA polymerase comprises T7 RNA polymerase.

10. Use of the dumbbell-shaped nucleic acid probe according to any one of claims 1 to 7 or the target detection kit based on the dumbbell-shaped nucleic acid probe according to claim 8 or 9 in nucleic acid detection or preparation of nucleic acid detection products.

11. A target detection method based on a dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes, characterized in that: include: (1) using the dumbbell-shaped nucleic acid probe according to any one of claims 1 to 7 or the target detection kit based on the dumbbell-shaped nucleic acid probe according to claim 8 or 9, adding a sample to be tested, and waiting for the dumbbell-shaped nucleic acid probe to form a circular template for rolling circle transcription; (2) performing rolling circle amplification transcription using the circular template of rolling circle transcription obtained in step (1); (3) Detection is performed using the rolling circle amplification transcription product obtained in step (2).

12. The target detection method based on dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes according to claim 11, characterized in that: When the dumbbell-shaped nucleic acid probe selects probe 1, probe 3 or probe 4: In step (1): the sample to be tested and the probe are added to a buffer solution to perform a first reaction; after the reaction, a ligation sequence and a ligase are added to the obtained reaction solution to perform a second reaction.

13. The target detection method based on dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes according to claim 12, characterized in that: The molar ratio of probe to linker sequence is 4-8:1-2; And / or, the first reaction is carried out at 10-70° C. for >2 min; And / or, the second reaction is carried out under the condition of incubating at 10-70° C. for >2 min.

14. The target detection method based on dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes according to claim 11, characterized in that: When the dumbbell-shaped nucleic acid probe selects probe 2: In step (1): the sample to be tested, the probe and the ligase are added to a buffer solution to carry out a reaction.

15. The target detection method based on dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes according to claim 14, The reaction conditions are 10-70° C. for >2 min.

16. The target detection method based on dumbbell-shaped nucleic acid probe for non-disease diagnosis or treatment purposes according to any one of claims 11 to 15, characterized in that: In step (2): adding a reaction mixture containing T7 RNA polymerase, NTP and buffer to the reaction product of step (1), and then reacting; optionally, the reaction condition is reacting at 10-70° C. for >2 min; And / or, in step (3), the rolling circle amplification transcription product obtained in step (2) is used to detect the CRISPR-Cas system; optionally, an activation sequence, a reporter gene, a Cas protein and a buffer are added to the reaction product of step (2) to react; optionally, the molar ratio of the activation sequence, the reporter gene and the Cas protein is in the range of 1000-2000:200-400:50-100; optionally, the reaction conditions are reaction at 10-70°C for >2 min.

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