A closed DNA triangle for visual detection of nucleic acids, a kit and a detection method

Abstract

The application discloses a closed DNA triangle for visual detection of nucleic acid, a kit and a detection method. The closed DNA triangle takes a DNA triangle as a linking core, and a pair of complementary single-base tandem primer chains are connected to the sticky ends of each of the three corners of the DNA triangle. The nucleic acid amplification reaction of the pair of single-base tandem primer chains can trigger the system to generate a DNA hydrogel. In the absence of a nucleic acid target, the pair of single-base tandem primer chains are correspondingly closed by a blocking chain Blocker1 and a blocking chain Blocker2. The sequence of the sticky end is the same as the sequence of the target nucleic acid sequence. The application outputs a specific signal through the unique phase transition from a liquid state to a gel state based on the pathogen nucleic acid triggered reaction system, and can avoid the color blindness and color weakness population from being unable to observe the colorimetric detection result or the fluorescence detection needing the aid of instruments.

Description

Technical Field

[0001] This invention relates to a closed DNA triangle, specifically to a closed DNA triangle, a kit, and a detection method for nucleic acid visualization detection. Background Technology

[0002] Since American scientist Kary Mullis invented polymerase chain reaction (PCR) technology in 1985, it has become the gold standard for clinical sample testing due to its high specificity and sensitivity, and is widely used in clinical nucleic acid testing. However, the technology still has limitations: the operation steps are cumbersome, the testing cycle is long (usually more than 2 hours), the equipment cost is high, and it is highly dependent on professional technicians.

[0003] To simplify procedures and shorten reaction times, various isothermal nucleic acid amplification techniques have emerged, such as exponential amplification (EXPAR), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), and the most representative loop-mediated isothermal amplification (LAMP). LAMP has become an important technique in the field of pathogen detection due to its ease of operation, rapid reaction characteristics (reaction completed in 60 minutes), and high detection sensitivity. There are two common types of signal outputs in LAMP: ① Fluorescent signals, such as calcein and SYBR green I (the fluorescence of the system increases after amplification, but it is difficult to distinguish under natural light and usually requires the use of a fluorescence detector); ② Color changes of the reaction system under natural light, such as the addition of hydroxynaphthol blue (the solution color changes from blue to purple before and after amplification, which is difficult for colorblind or color-weak people to distinguish) (Stem-loop-primer assisted isothermal amplification enabling high-specific and ultrasensitive nucleic acid detection, Biosens. Bioelectron. 184(2021) 113239; Mathematical model to reduce loop mediated isothermalamplification (LAMP) false-positive diagnosis, Electrophoresis 40 (2019)2706–2717).

[0004] Furthermore, as the primary carrier of genetic information, the structure and functional regulation mechanisms of nucleic acids have always been a research hotspot. In 1982, Seeman and his colleagues constructed a grid-like DNA nanostructure by splicing together branched DNA with sticky ends, officially opening the door to DNA nanomaterials research (Shape-controllable synthesis of functional nanomaterials on DNA templates, Chemical Research in Chinese Universities, 36 (2020) 171−176). In recent years, supported and rapidly developed by chemical and biosynthetic technologies, DNA nanomaterials have become a research focus in fields such as biosensing, drug delivery, and cancer treatment.

[0005] To enter cells and tissues, micro DNA nanodevices or clusters have certain rigidity and size (0-300 nm) requirements. Common forms include branched, spherical, tetrahedral, tubular, sheet-like, and even multimorphic composite stacked structures, which can be classified as DNA origami or framework nucleic acids (Self-assembled DNA nanomaterials with highly programmed structures and functions, Materials Chemistry Frontiers, 2 (2018) 423−436; Functional aptamer-embedded nanomaterials for diagnostics and therapeutics, ACS Applied Materials & Interfaces, 13 (2021) 9542−9560; Designer nanomaterials through programmable assembly, Angewandte Chemie-International Edition, 61 (2022) e202105678). In addition, DNA hydrogels, novel nucleic acid nanocarriers with macroscopic dimensions, have attracted widespread attention and research interest in the field of clinical diagnosis and treatment in recent years due to their good biocompatibility, manipulability and high loading capacity.

[0006] From the perspective of DNA hydrogel preparation, nucleic acid amplification of tandem repeats is a common method for preparing DNA hydrogels. Currently, rolling circle amplification (RCA) technology is widely used (Research Progress on Rapid Gel Formation Strategies for DNA Hydrogels Based on RCA Products, Analytical Laboratory, 44 (2025), 462–468). First, a circular DNA strand is constructed as a template; then, pre-designed primers are added, and a product containing tandem repeats is amplified with the participation of a DNA polymerase with strand displacement activity (such as Phi 29). Finally, the amplified product is concentrated and annealed to prepare a homozygous DNA hydrogel. RCA preparation of DNA hydrogels faces the following challenges in its widespread application: ① The operation is cumbersome and time-consuming: RCA requires the construction of a circular template, and Phi 29 has low polymerization activity, often requiring overnight or even several days for amplification to complete; ② The nucleic acid amplification product is linear DNA, which is difficult to directly form a cross-linked network structure to form a hydrogel, often requiring further purification, concentration, and annealing of the amplified product.

[0007] From the perspective of nucleic acid detection, there are two common signal output methods for detection results such as PCR and nucleic acid isothermal amplification: ① Calcein and SYBR Green I, the fluorescence of the system is enhanced after amplification, but it is difficult to distinguish under natural light and usually requires the use of a fluorescence detector; ② Hydroxynaphthol blue, the solution color changes from blue to purple before and after amplification, which is not easy for colorblind or color-weak people to distinguish.

[0008] Therefore, developing a simple, rapid, and easy-to-interpret nucleic acid detection method and a corresponding DNA hydrogel preparation technology is of great practical significance.

[0009] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0010] The purpose of this invention is to provide a closed DNA triangle, a reagent kit, and a detection method for nucleic acid visualization detection. This invention solves the problems of cumbersome operation steps, long detection cycle, and difficulty in distinguishing detection results under natural light or being unfriendly to colorblind or color-weak individuals in the prior art. This invention outputs specific signals through a unique phase transition ("liquid to gel state") of a reaction system triggered by pathogen nucleic acid, which can avoid the difficulties of colorblind or color-weak individuals being unable to observe colorimetric detection results or the need for instrument assistance for fluorescence detection.

[0011] To achieve the above objectives, the present invention provides a closed DNA triangle for nucleic acid visualization detection. This closed DNA triangle uses a DNA triangle as its connecting core, and at each of the three corners of the DNA triangle, a pair of complementary single-base tandem primer strands are attached to their sticky ends. These single-base tandem primer strands are respectively closed by blocking strands Blocker1 and Blocker2. The sequence of the sticky ends is partially identical to the target nucleic acid sequence. The sequence of Blocker1 consists of a sequence complementary to the target nucleic acid and a sequence complementary to the single-base tandem primer strand. The sequence of Blocker2 consists of a sequence complementary to the target nucleic acid and a sequence complementary to the other single-base tandem primer strand.

[0012] The closed DNA triangle B-DT designed in this invention is a highly stable DNA self-assembly structure, consisting of a main body, three grafted pairs of complementary single-base tandem primer chains, and a blocking strand. Its stability stems from the DNA triangle main structure and the effective blocking of the single-base tandem primer chains by the blocking strand. Under normal conditions, due to the presence of the blocking strand, the single-base tandem primer chains cannot freely participate in the nucleic acid amplification reaction, keeping the entire system in a liquid phase. When the target nucleic acid is present in the sample to be tested, the target nucleic acid will complementaryly pair with the blocking strands Blocker1 and Blocker2, thereby releasing DT. DT initiates the nucleic acid amplification reaction under the action of Bst DNA polymerase, dNTPs, and amplification buffer. As the amplification reaction proceeds, A12 and T12 grafted onto the DNA triangle amplify to form a tight 3D network structure, ultimately causing a phase transition from liquid to gel. Based on the signal output of the reaction system transitioning from a liquid state before amplification to a gel state after amplification, this invention proposes a nucleic acid target-triggered in situ DNA hydrogel preparation strategy for visual detection (NHV). This method, which outputs a specific signal based on the phase transition of the reaction system triggered by pathogen nucleic acid, eliminates the need for complex fluorescence detection instruments. The presence of target nucleic acid in a sample can be intuitively determined by observing changes in the solution state under natural light. This avoids the difficulties faced by colorblind or color-weak individuals in observing colorimetric detection results or the need for instrumental assistance in fluorescence detection, greatly simplifying the nucleic acid detection process, shortening the detection cycle, and demonstrating significant application advantages.

[0013] Preferably, the pair of complementary single-base tandem primer chains are primer chains composed of tandem A bases and tandem T bases, respectively, and are at least 8 bases in length. A8 (AAAAAAAA, 8 nt) and longer sequences, T8 (TTTTTTTT, 8 nt) and longer sequences can trigger rapid isothermal amplification.

[0014] Preferably, the DNA triangle is prepared from its backbone nucleotide sequence D7 and single strands D1-D6; wherein, the sequence of single strand D1 consists of a sequence complementary to the single strand D5 portion + a sequence complementary to the backbone nucleotide sequence D7 portion + a sequence complementary to the single strand D4 portion + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; the sequence of single strand D2 consists of a sequence complementary to the single strand D4 portion + a sequence complementary to the backbone nucleotide sequence D7 portion + a sequence complementary to the single strand D6 portion + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; the sequence of single strand D3 consists of a sequence complementary to the single strand D6 portion + a sequence complementary to the backbone nucleotide sequence D7 portion + a sequence complementary to the single strand D5 portion + a sequence complementary to the backbone nucleotide sequence D6 portion + a sequence complementary to the target nucleic acid sequence + a single-base tandem primer sequence; The sequence of a single-stranded D4 consists of a complementary sequence to the single-stranded D5 portion, a sequence partially identical to the target nucleic acid sequence, and a single-base tandem primer chain. The sequence of a single-stranded D4 consists of a complementary sequence to the single-stranded D1 portion, a complementary sequence to the single-stranded D2 portion, a sequence partially identical to the target nucleic acid sequence, and another single-base tandem primer chain. The sequence of a single-stranded D5 consists of a complementary sequence to the single-stranded D3 portion, a complementary sequence to the single-stranded D1 portion, a sequence partially identical to the target nucleic acid sequence, and another single-base tandem primer chain. The sequence of a single-stranded D6 consists of a complementary sequence to the single-stranded D2 portion, a complementary sequence to the single-stranded D3 portion, a sequence partially identical to the target nucleic acid sequence, and another single-base tandem primer chain.

[0015] Preferably, the DNA triangle has its backbone nucleotide sequence D7 as shown in SEQ ID NO.7.

[0016] More preferably, the sequence of single-stranded D1 is: the sequence shown in SEQ ID NO. 12 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; the sequence of single-stranded D2 is: the sequence shown in SEQ ID NO. 13 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; the sequence of single-stranded D3 is: the sequence shown in SEQ ID NO. 14 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; the sequence of single-stranded D4 is: the sequence shown in SEQ ID NO. 15 + a sequence partially identical to the target nucleic acid sequence + another single-base tandem primer sequence; the sequence of single-stranded D5 is: the sequence shown in SEQ ID NO. 16 + a sequence partially identical to the target nucleic acid sequence + another single-base tandem primer sequence; the sequence of single-stranded D6 is: the sequence shown in SEQ ID NO. 17 + a sequence partially identical to the target nucleic acid sequence + another single-base tandem primer sequence.

[0017] A second objective of the present invention is to provide a kit containing the aforementioned closed DNA triangle.

[0018] Preferably, the kit further comprises: a nucleic acid polymerase with strand displacement activity, dNTPs, and an amplification buffer solution.

[0019] More preferably, the nucleic acid polymerase with strand displacement activity is selected from Bst DNA polymerase. The reaction temperature is set according to the selected DNA polymerase; the optimal reaction temperature for Bst DNA polymerase is 65°C. If Bst DNA polymerase is selected, the reaction temperature can be set to [specific temperature range].

[0020] More preferably, the dNTP is selected from dATP and dTTP; or / and the amplification buffer solution comprises: 20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 5 mM MgSO4 and 0.1% Tween-20, pH 8.8.

[0021] A third objective of this invention is to provide a nucleic acid visualization detection method based on in-situ DNA hydrogel preparation triggered by nucleic acid targets. This method comprises: incubating the sample to be tested and the described closed DNA triangle in a solution containing a strand displacement-active nucleic acid polymerase, dNTPs, and an amplification buffer at a suitable temperature for the strand displacement-active nucleic acid polymerase; observing the solution state or quantifying the target nucleic acid in the sample to be tested by detecting fluorescence intensity; if the solution gels, it contains the target nucleic acid.

[0022] Preferably, the incubation reaction time is 30 minutes or more.

[0023] Preferably, the concentration of the closed DNA triangle is above 50 nM.

[0024] The closed DNA triangle, reagent kit, and detection method for nucleic acid visualization detection of the present invention solve the problems of cumbersome operation steps, long detection cycle, and difficulty in distinguishing detection results under natural light or being unfriendly to people with color blindness or color weakness in the prior art. It has the following advantages: (1) This invention has carefully designed a DNA triangle (B-DT) containing a “blocked DNA strand”. At 65°C, the target nucleic acid (such as human immunodeficiency virus, HIV) can bind to the “blocked strand” and remove it from the B-DT, thereby activating the A12 and T12 on the DT to undergo ultra-rapid isothermal amplification, so that the reaction solution changes from a solution state to a gel state. For colorblind, color weak and elderly people, the gelation signal output is easier to identify;

[0025] (2) The in-situ rapid preparation strategy of DNA hydrogel based on nucleic acid amplification developed in this invention has been successfully applied to nucleic acid detection. The NHV detection method developed in this way can detect pathogen nucleic acid in 30 min without the need for detection instruments, and the detection sensitivity can reach 0.5 copies / μL.

[0026] (3) Compared with the existing fluorescence and solution color change signal output methods of PCR and LAMP, the method of the present invention achieves naked-eye visual interpretation through solid-liquid phase change (gelation) of reaction solution, without relying on fluorescence detection equipment or complex color development system. Moreover, the gelation results can be stored for a long time, have strong resistance to environmental interference, and are convenient for long-term verification. (4) Compared with PCR and LAMP, the method of the present invention is simpler to operate, is a one-step operation, is simple to design, and only requires changing the base sequence of the closed strand and the corresponding part of DT for different targets. Moreover, it does not require an expensive thermal cycling fluorescence detector, and only needs to observe whether the reaction solution is gelled. Attached Figure Description

[0027] Figure 1 The diagram illustrates the principle of isothermal amplification of nucleic acids to prepare DNA hydrogels according to the present invention; (A) A12 and T12 amplification principle; (B) DNA triangle (DT) amplification principle containing A12 and T12.

[0028] Figure 2 For the feasibility verification of DT amplification of the present invention and the optimization results of A12 and T12, DT amplification conditions; (A) A12+T12, DT amplification; (B) Amplification with different A12 and T12 concentrations; (C) Amplification with different DT concentrations; (D) DT amplification at different times.

[0029] Figure 3 The structural characterization of the hydrogels amplified in this invention includes: (A) A viscous hydrogel formed by A12 and T12; (B) Hydrogel formed by DT containing A12 and T12; (C) Atomic force microscopy characterization of the amplified products of A12 and T12; (D) Atomic force microscopy characterization of the amplified products of DT containing A12 and T12; (E) Scanning electron microscopy characterization of the viscous hydrogel formed by A12 and T12; and (F) Scanning electron microscopy characterization of the hydrogel formed by DT containing A12 and T12.

[0030] Figure 4 The following are the principles of NHV detection in this invention: (A) Preparation of B-DT; (B) Demonstration of NHV detection principle; (C) Detailed structure of DT.

[0031] Figure 5 The results are the NHV detection capability verification results (A) and clinical sample verification results (B) of this invention. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0034] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0035] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0036] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0037] This invention provides a closed DNA triangle, a reagent kit, and a detection method for nucleic acid visualization detection. Based on the grafting of single-base tandem primer pairs A12 and T12 onto a DNA triangle (DT) module, using DT as the binding core, it enables rapid isothermal amplification and in-situ preparation of DNA hydrogels without the need for a template (see [link to original text]). Figure 1 This invention designs a closed DNA triangle B-DT, which is a highly stable DNA self-assembly structure (see...). Figure 4 Under normal conditions, due to the presence of the blocking strand, the single-base tandem primer chains cannot freely participate in the nucleic acid amplification reaction, keeping the entire system in a liquid phase. When the target nucleic acid is present in the sample to be tested, the target nucleic acid will complementaryly pair with the blocking strands Blocker1 and Blocker2, thereby releasing DT. DT initiates the nucleic acid amplification reaction under the action of Bst DNA polymerase, dNTPs, and amplification buffer. As the amplification reaction proceeds, A12 and T12 grafted onto the DNA triangle amplify to form a tight 3D network structure, ultimately causing a phase transition from liquid to gel.

[0038] like Figure 1The diagram illustrates the principle of DNA hydrogel preparation via ultrafast isothermal amplification of nucleic acids according to this invention. Double-stranded DNA exhibits a dynamic equilibrium between unwinding and renaturation, a phenomenon known as DNA respiratory fluctuation. The isothermal amplification products used in this invention are all AT base-paired, with low Tm values ​​and strong respiratory fluctuations. First, A12 and T12 hybridize at arbitrary lengths to form primer dimers (step 1). Extension using Bst enzyme (step 2) lengthens the DNA double strand. Due to respiratory fluctuations, the ends of the strand are easily in an unwound, open state, readily hybridizing with other primer dimers or their extension products (step 3a). With the participation of Bst enzyme, further extension occurs, replacing the complementary strand (step 4a). Simultaneously, the replaced complementary strand also hybridizes and extends (step 5a), a phenomenon known as respiratory amplification. Similarly, due to highly repetitive sequences, regional mismatches easily occur during renaturation of the DNA strand (step 3b). DNA polymerase uses dATP and dTTP to fill these mismatches, also leading to DNA strand elongation (steps 4b and 5b). This phenomenon is classic sliding amplification. This invention grafts A12 and T12 onto the 3' end of a DNA triangular nanostructure, as follows: Figure 1 As shown in B. Due to its good thermal stability and rigidity, DT can act as a network connection center in nucleic acid amplification, accelerating the formation of a tight network structure to lock in water molecules in the reaction product system, ultimately causing the reaction system to change from a liquid state to a gel state.

[0039] The following embodiments provide a detailed description of the closed DNA triangle, reagent kit, and detection method for nucleic acid visualization detection provided by the present invention.

[0040] Some of the reagents, instruments, and sequences used in the following examples are shown below: 1. Materials and reagents Deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), and Bst DNA polymerase were provided by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). All oligonucleotides were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are listed in Table 1. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), ammonium sulfate ((NH4)2SO4), potassium chloride (KCl), magnesium sulfate (MgSO4), and Tween-20 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ultrapure water (≥18.2 M Ω·cm) was purified using a water purification system (Ulupure Ultratech Co., Ltd.) and used in all experiments.

[0041] The amplification buffer contains: 20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 5 mM MgSO4 and 0.1% Tween-20, pH 8.8.

[0042] 2. Instruments The detection performance of the NHV reaction was validated on a Dhelix-1610 isothermal fluorescence detector (Double Helix Gene Technology Co., Ltd.). Gel electrophoresis images were scanned using a Fusion Fx vilber lourmat imaging system (vilber). Atomic force microscopy (AFM) images were recorded by a Dimension Icon (Brook) using its tapping mode at its resonant frequency. Scanning electron microscopy (SEM) images were recorded by a thermal field emission Tescom CLARA (Tescom (China) Co., Ltd.).

[0043] 3. Amplification primer and probe sequences Table 1 lists the amplification primer and probe sequences used in the following examples. Note: The sequences under the wavy lines in D1~D6 are numbered sequentially as SEQ ID NO.12~17; the sequences in Blocker1 and Blocker2 in Table 1 are designed using the HIV-1 gene as an example. If other nucleic acids are tested, the sequences of Blocker1 and Blocker2 can be designed according to the target nucleic acid (single strand). The Blocker1 and Blocker2 sequences in Table 1 are partially paired with the target nucleic acid HIV-1 gene sequence.

[0044] Example 1: Preparation of DNA Hydrogel 1. Preparation of DNA hydrogels based on A12 and T12 Mix 50 nM single-base tandem primer A12 and 50 nM single-base tandem primer T12 with amplification buffer containing 70 U / mL BstDNA polymerase, 1.25 mM dATP and 1.25 mM dTTP, and incubate at 65°C for 30 minutes.

[0045] 2. Preparation of DNA hydrogels based on DNA triangles containing A12 and T12 (1) Preparation of DNA triangles containing A12 and T12 Mix 2 μM of D1~D7 into the amplification buffer solution, anneal at 95℃ for 5 minutes, allow to stand at room temperature for 2 hours before use, and record as DT; (2) Preparation of DNA hydrogels The 50 nM DNA triangles prepared above were incubated with the above amplification buffer containing 70 U / mL Bst DNA polymerase, 1.25 mM dATP and 1.25 mM dTTP at 65°C for 10 minutes.

[0046] Example 2: Feasibility Verification of DT Amplification To verify the feasibility of preparing DNA hydrogels in Example 1 above, the preparation of DNA hydrogels based on DNA triangles containing A12 and T12 was the same as in Example 1. The preparation of DNA hydrogels based on A12 and T12 was basically the same as in Example 1, except that the concentrations of A12 and T12 were both 150 nM.

[0047] The amplification products were sonicated and diluted 10-fold, then mixed with 6-fold glycerol gel loading buffer. 10 μL of each mixture was added to a 1% agarose gel (wt%) for electrophoresis. The gels were electrophoresed at 110 V in 1-fold TAE buffer at room temperature for 50 minutes, and then imaged using a Fusion FX Vilber Lourmat imaging system.

[0048] like Figure 2 As shown in A, this is the result of the feasibility verification of DT amplification in Example 1 of the present invention. Lane 1 is the amplification result of A12+T12. It can be seen that A12 and T12 produce a large number of high molecular weight DNA chains. Lane 2 is the unamplified DT. Lane 3 is the amplification result of DT. It can be seen that the DNA triangle containing A12 and T12 also has a large number of high molecular weight products.

[0049] Furthermore, the structures of the hydrogels prepared from A12 and T12, as well as the hydrogel prepared from DT, were characterized. The results are shown in [link to relevant documentation]. Figure 3 .

[0050] like Figure 3 Figure A shows a photograph of the hydrogels prepared from A12 and T12. It can be seen that the hydrogels are liquid at 0 min and become viscous hydrogels after 30 min of incubation. Figure 3 As shown in Figure B, this is a photograph of the hydrogel prepared by DT. Since the DNA triangle acts as a linker center during the amplification process, it tightly connects the DNA long chain formed by A12 and T12. At 65°C, 50 nM DT amplification takes about 10 minutes to form a hydrogel that precipitates out of the reaction system and can be picked up directly from the test tube. The yield can reach 70% (wt%).

[0051] like Figure 3 Figure C shows the AFM characterization results of hydrogels prepared by A12 and T12 after an incubation time of 5 min. Figure 3 Figure D shows the AFM characterization results of the hydrogel prepared by DT with an incubation time of 5 min. (Comparison) Figure 3 As can be seen from C and D, DT can increase the local concentration of amplification products and form a large number of network structures, which is beneficial for the in-situ preparation of DNA hydrogels.

[0052] like Figure 3 Figure E shows the SEM characterization results of the hydrogels prepared from A12 and T12. Figure 3 Figure F shows the SEM characterization results of the hydrogels prepared by DT. It can be seen that the hydrogels prepared by A12, T12, and DT all have the typical loose and porous structure of DNA hydrogels.

[0053] Example 3 Optimization of DT Amplification Reaction Conditions 1. Effect of different substrate concentrations on the preparation of DNA hydrogels To investigate the effect of different concentrations on amplification, different concentrations of A12 and T12 (100, 150, 500, 700, and 1000 nM) were used for amplification in the reaction of preparing DNA hydrogels based on A12 and T12 in Example 1. Different concentrations of DT (10, 30, 50, 70, and 100 nM) were also used for the reaction of preparing DNA hydrogels based on DT in Example 1. Since DNA triangles act as linking centers in amplification, a small amount of DNA triangles can be amplified to achieve rapid preparation of DNA hydrogels. If the concentration of DNA triangles is too high, it will inhibit the amplification reaction. Therefore, the concentration designed for the reaction of preparing DNA hydrogels based on DT in Example 1 was relatively small. The system was amplified and subjected to agarose gel electrophoresis to explore the optimal DNA concentration for the two gel preparation methods.

[0054] like Figure 2 Figure B shows the results of amplification experiments with different A12 and T12 concentrations. It can be seen that the product amount gradually increases with increasing A12 and T12 concentrations. Figure 2 Figure C shows the amplification results of different DT concentrations. It can be found that a higher yield is achieved when the DT concentration is 50 nM.

[0055] 2. Effects of different time points on the preparation of DNA hydrogels To investigate the effect of different incubation times on amplification, the reaction of DNA hydrogel prepared based on DT in Example 1 was amplified with different incubation times and subjected to agarose gel electrophoresis.

[0056] like Figure 2 As shown in Figure D, the experimental results are obtained for different DT amplification times. It can be seen that under the condition of reaction at 65℃ for 10 min, 50 nM DT can produce a large number of amplification products.

[0057] Example 4: Preparation of a closed DNA triangle A closed DNA triangle (B-DT) for nucleic acid visualization detection, the preparation method of which includes: Mix 2 μM D1~D7 with 6 μM Blocker1 and Blocker2 in amplification buffer solution, anneal at 95°C for 5 minutes, allow to return to room temperature, and let stand for 2 hours before use.

[0058] Example 5: NHV Detection Method To apply DT amplification to target nucleic acid detection, this invention proposes an in-situ preparation method for nucleic acid target-triggered DNA hydrogels for nucleic acid visualization detection technology (NHV). Specifically, the closed DNA triangles prepared in Example 4 are used for NHV detection, as detailed below: Add 5 μL of target nucleic acid sample to 95 μL of amplification buffer solution containing 50 nM B-DT, 70 U / mL Bst DNA polymerase, 1.25 mM dATP, and 1.25 mM dTTP, and react at 65℃ for 30 min. Gel formation of the solution can be observed.

[0059] like Figure 4 The diagram illustrates the NHV detection principle of this invention. Figure 4 A is B-DT, prepared by annealing DT with Blocker sequences. DT contains 3 A12 and 3 T12 sequences. Blocker1 and Blocker2 block T12 and A12 respectively. The blocking strand is a sequence complementary to the nucleic acid target region, used to inhibit DT amplification. For a detailed structural representation of the prepared blocked DNA triangle, see [link to diagram]. Figure 4 C. Figure 4 The B-DT diagram illustrates the principle of NHV detection. With the replacement of the nucleic acid target, Blocker1 and Blocker2 of B-DT trigger DT amplification, transforming the reaction solution from a solution state to a gel state. Conversely, if there is no nucleic acid target, the Blockers inhibit DT amplification activity, and the reaction solution state remains unchanged.

[0060] Example 6: One-step NHV method for detecting HIV nucleic acid 1. Construct a plasmid containing the HIV-1 gene. The HIV-1 gene sequence information was obtained from the GenBank database. The plasmid contained the first 300 bp of the HIV-1 gene AF033819.3. The gene fragment was synthesized and inserted into the pUC57 plasmid. Samples containing different plasmid DNA concentrations (0, 0.5, 5, 50, 500, 5000, and 50000 copies / μL) were prepared, and the plasmid DNA concentration was determined using a Multiskan Go microplate spectrophotometer (Thermo Scientific, MA, USA).

[0061] The formula for calculating plasmid DNA copy number is as follows: DNA copy number = (ng × 6.02 × 10) 23 × 10 −9 () / (fragment length [bp] × 660) 2. HIV nucleic acid detection using the NHV method. The detection process for NHV is very simple; the nucleic acid sample is simply added to the amplification system, as detailed below: Add 5 μL of the above-prepared sample containing plasmid DNA to 95 μL of amplification buffer solution containing (50 nM B-DT), 70 U / mL BstDNA polymerase, 1.25 mM dATP, and 1.25 mM dTTP. Incubate at 65℃ for 30 min and observe the changes in solution state. If precise quantification is required, add SYBR Green I (1 / 10000 dilution) to the reaction system and detect the fluorescence intensity using a constant temperature fluorescence detector.

[0062] 3. Experimental Results like Figure 5 As shown in Figure A, the detection results of the plasmid containing the HIV fragment of this invention can be observed. It can be seen that as the target nucleic acid concentration increases, the amplification curve appears earlier and earlier, indicating that specific amplification has occurred, but all can be initiated within 15 minutes. According to experimental observations, unlike DT, which only requires 10 minutes to directly prepare the hydrogel, the NHV system requires approximately 30 minutes for gelation, and the detection limit can reach 0.5 copies / μL.

[0063] Example 7: Clinical Sample Validation To further verify the reliability and practicality of the NHV detection method of this invention, clinical samples (one without HIV-1 and three containing HIV-1) were selected for testing. The test results are shown below. Figure 5 B, like Figure 5 As shown in Figure B, clinical sample validation revealed that only positive clinical samples triggered DNA hydrogel formation, and the product remained firmly attached to the tube wall even after the reaction tube was inverted. Negative samples did not trigger hydrogel preparation, and the reaction solution flowed to the bottom of the inverted tube. This indicates that the NHV detection method can effectively distinguish the presence of HIV-1 nucleic acid in clinical samples, without false positives or false negatives, and has good potential for clinical application.

[0064] The above results all indicate that, through the careful design of B-DT, the present invention provides NHV with excellent response sensitivity to nucleic acid targets, and the detection time of 30 min is much shorter than that of typical PCR technology (about 2 h).

[0065] In summary, the nucleic acid target-triggered in-situ DNA hydrogel preparation proposed in this invention is used for nucleic acid visualization detection technology. Its unique closed DNA triangle design achieves highly sensitive detection of nucleic acid targets. This method is not only simple to operate, requiring only the direct addition of nucleic acid samples to the amplification system without complex pretreatment steps, but also has a short detection time, completing the detection within 30 minutes, significantly shorter than traditional PCR technology, providing strong support for rapid clinical diagnosis.

[0066] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A closed DNA triangle for nucleic acid visualization detection, characterized in that, The closed DNA triangle uses a DNA triangle as the core of the connection. At the sticky end of each of the three corners of the DNA triangle, there is a pair of complementary single-base tandem primers. The pair of single-base tandem primers are respectively closed by blocking strands Blocker1 and Blocker2. Wherein, the sequence of the sticky ends is a sequence that is partially identical to the target nucleic acid sequence; The sequence of the closed strand Blocker1 consists of a sequence that is complementary to the target nucleic acid portion and a single-base tandem primer strand that is complementary to the target nucleic acid portion. The sequence of the closed-chain Blocker2 consists of a sequence complementary to the target nucleic acid portion and another single-base tandem primer sequence complementary to each other.

2. The closed DNA triangle according to claim 1, characterized in that, The pair of complementary single-base tandem primer chains, consisting of A bases and T bases respectively, are at least 8 bases in length.

3. The closed DNA triangle according to claim 1, characterized in that, The DNA triangle is prepared from its backbone nucleotide sequence D7 and single strands D1~D6; The sequence of the single-stranded D1 consists of a sequence complementary to the single-stranded D5 portion, a sequence complementary to the backbone nucleotide sequence D7 portion, a sequence complementary to the single-stranded D4 portion, a sequence partially identical to the target nucleic acid sequence, and a sequence of a single-base tandem primer chain. The sequence of the single-stranded D2 consists of a sequence complementary to the single-stranded D4 portion, a sequence complementary to the backbone nucleotide sequence D7 portion, a sequence complementary to the single-stranded D6 portion, a sequence identical to the target nucleic acid sequence portion, and a sequence of a single-base tandem primer chain. The sequence of the single-stranded D3 consists of a sequence complementary to the single-stranded D6 portion, a sequence complementary to the backbone nucleotide sequence D7 portion, a sequence complementary to the single-stranded D5 portion, a sequence identical to the target nucleic acid sequence portion, and a sequence of a single-base tandem primer chain. The sequence of the single-stranded D4 consists of a sequence complementary to the single-stranded D1 portion, a sequence complementary to the single-stranded D2 portion, a sequence partially identical to the target nucleic acid sequence, and the sequence of another single-base tandem primer chain. The sequence of the single-stranded D5 consists of a sequence complementary to the single-stranded D3 portion, a sequence complementary to the single-stranded D1 portion, a sequence partially identical to the target nucleic acid sequence, and the sequence of another single-base tandem primer chain. The sequence of the single-stranded D6 consists of a sequence complementary to the single-stranded D2 portion, a sequence complementary to the single-stranded D3 portion, a sequence partially identical to the target nucleic acid sequence, and the sequence of another single-base tandem primer chain.

4. The closed DNA triangle according to any one of claims 1 to 3, characterized in that, The DNA triangle is composed of its backbone nucleotide sequence D7 as shown in SEQ ID NO.

7.

5. The closed DNA triangle according to claim 4, characterized in that, The sequence of the single-stranded D1 is: the sequence shown in SEQ ID NO. 12 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; The sequence of the single-stranded D2 is: the sequence shown in SEQ ID NO.13 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; The sequence of the single-stranded D3 is: the sequence shown in SEQ ID NO.14 + a sequence partially identical to the target nucleic acid sequence + a single-base tandem primer sequence; The sequence of the single-stranded D4 is: the sequence shown in SEQ ID NO.15 + a sequence partially identical to the target nucleic acid sequence + the sequence of another single-base tandem primer chain; The sequence of the single-stranded D5 is: the sequence shown in SEQ ID NO.16 + a sequence partially identical to the target nucleic acid sequence + the sequence of another single-base tandem primer chain; The sequence of the single-stranded D6 is: the sequence shown in SEQ ID NO.17 + a sequence partially identical to the target nucleic acid sequence + the sequence of another single-base tandem primer chain.

6. A kit containing the closed DNA triangle as described in any one of claims 1 to 5.

7. The reagent kit according to claim 6, characterized in that, The kit also contains: a nucleic acid polymerase with strand displacement activity, dNTPs, and an amplification buffer solution.

8. The reagent kit according to claim 6, characterized in that, The dNTPs are selected from dATP and dTTP; Or / and, the amplification buffer solution comprises: 20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 5 mM MgSO4 and 0.1% Tween-20, pH 8.

8.

9. A method for visual detection of nucleic acids based on in-situ preparation of DNA hydrogels triggered by nucleic acid targets, characterized in that, The method includes: The sample to be tested and the closed DNA triangle as described in any one of claims 1 to 5 are incubated in a nuclease with strand displacement activity, dNTPs and an amplification buffer solution at the applicable temperature of the nuclease with strand displacement activity. The solution state is observed or the target nucleic acid in the sample to be tested is quantified by detecting the fluorescence intensity. If the solution gels, it contains the target nucleic acid.

10. The nucleic acid visualization detection method according to claim 9, characterized in that, The incubation reaction time is more than 30 minutes.

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