Kit and detection method capable of detecting different types of targets based on DNA multi-pass knots

Abstract

The invention relates to a kit capable of detecting different types of targets based on DNA multi-pass knots and a detection method. The kit comprises a detection composition and a test strip, the detection composition comprises a molecular beacon and an auxiliary chain, wherein the nucleotide sequence of the molecular beacon (MB) is as shown in SEQ ID No.1; the auxiliary chain is a combination of HBV-alpha1 and HBV-beta1, and / or a combination of miR-21-alpha2 and miR-21-beta2, and / or a combination of ATP-alpha3 and ATP-beta3, and / or a combination of Thrombin-alpha4 and Thrombin-beta4, and / or a combination of TdT-alpha5 and TdT-beta5, and the sequence of the auxiliary chain is as shown in SEQ ID No.2-11. The kit can detect different types of targets, and when a target exists, no matter DNA / RNA, small molecules, proteins or enzymes, alpha and beta auxiliary chains can be connected together and hybridized with MB to form a DNA multi-pass node, so that a test line develops color, the target is conveniently and intuitively determined, and the detection time is shortened. Remodification of nano-particles, re-pretreatment of a pad / film and assembly and cutting of test paper are avoided, and a large amount of manpower, material resources and financial resources are saved.

Description

Technical Field

[0001] The invention relates to a kit and a detection method for detecting different types of targets based on DNA multi-pass junctions, and belongs to the technical field of biological detection. Background Art

[0002] HBV is a DNA virus belonging to the Hepadnaviridae family that can cause chronic hepatitis, which can progress to cirrhosis or hepatocellular carcinoma. MiR-21 is a miRNA abundantly expressed in mammalian cells. Under physiological conditions, it regulates cell differentiation, apoptosis, and homeostasis. Its overexpression in human cells has been linked to cellular carcinogenesis. The small biological molecule adenosine 5'-triphosphate (ATP) is the universal energy currency in organisms, providing energy for various intracellular physiological activities and also serving as a signaling molecule for intercellular communication and immune regulation. Thrombin is a multifunctional serine protease and a core regulator of the coagulation process. Dysregulation of thrombin activity not only exacerbates thrombotic disorders but also triggers inflammatory cascades, neurodegenerative diseases, and cancer metastasis. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase whose expression is elevated in patients with leukemias such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML).

[0003] DNA multijunctions are structurally composed of multiple branched helical arms. In DNA nanotechnology, multijunctions have been used as a linker for branched connections to construct more complex structures. Due to their design flexibility, multijunctions can be used for detection. During detection, a molecular beacon is typically activated or its assembly conformation is altered, and fluorescence is restored during the DNA multijunction formation process with the assistance of a target.

[0004] Lateral flow analysis is a paper-based analytical method in which the sample moves across a membrane via capillary action. It typically consists of a sample pad containing the sample solution, a conjugate pad coated with DNA-modified gold nanoparticles (AuNPs), a nitrocellulose membrane with test and control lines, and an absorbent pad to absorb excess reagents and prevent backflow. As the sample solution flows, the pre-immobilized reagents hydrate, activating and identifying the target. As the solution flows, AuNPs gradually accumulate and stain the test line.

[0005] Lateral flow analysis requires no equipment, is easy to carry, and has the advantages of simple operation, visual detection, and stable storage. It can be used to easily detect targets. However, one type of test paper is usually only used to detect one type of target. In the process of making the test paper, gold nanoparticles need to be modified with specific oligonucleotides or antibodies, and corresponding capture units are set on the test line and control line. When the target changes, they need to be changed, redesigned, and reprepared. In addition, the sample pad needs to be pretreated, and the conjugated pad and nitrocellulose membrane need to be sprayed with modified AuNPs and capture units, respectively. These pretreatment processes, as well as the subsequent pad drying, overall assembly, and test paper cutting, need to be repeated for another target.

[0006] Therefore, there is an urgent need to provide a simple, rapid, sensitive, and specific kit and detection method that can detect different types of targets. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the present invention provides a kit and a detection method based on DNA multi-junction that can detect different types of targets.

[0008] The technical solutions of the present invention are as follows:

[0009] A detection composition based on DNA multi-junction, comprising a molecular beacon and an auxiliary chain;

[0010] The nucleotide sequence of the molecular beacon (MB) is shown in SEQ ID No. 1;

[0011] The auxiliary chain is a combination of HBV-α1 and HBV-β1, and / or a combination of miR-21-α2 and miR-21-β2, and / or a combination of ATP-α3 and ATP-β3, and / or a combination of Thrombin-α4 and Thrombin-β4, and / or a combination of TdT-α5 and TdT-β5;

[0012] The nucleotide sequence of the HBV-α1 is shown in SEQ ID No. 2;

[0013] The nucleotide sequence of the HBV-β1 is shown in SEQ ID No. 3;

[0014] The nucleotide sequence of miR-21-α2 is shown in SEQ ID No. 4;

[0015] The nucleotide sequence of miR-21-β2 is shown in SEQ ID No. 5;

[0016] The nucleotide sequence of the ATP-α3 is shown in SEQ ID No. 6;

[0017] The nucleotide sequence of the ATP-β3 is shown in SEQ ID No. 7;

[0018] The nucleotide sequence of Thrombin-α4 is shown in SEQ ID No. 8;

[0019] The nucleotide sequence of Thrombin-β4 is shown in SEQ ID No.9;

[0020] The nucleotide sequence of TdT-α5 is shown in SEQ ID No. 10;

[0021] The nucleotide sequence of TdT-β5 is shown in SEQ ID No.11.

[0022] Preferably, according to the present invention, the target detected by the detection composition is hepatitis B virus (HBV), miR-21, ATP, thrombin or terminal deoxynucleotidyl transferase (TdT); the target gene for detecting HBV-α1 and HBV-β1 is shown in SEQ ID No.12, and the target gene for detecting miR-21-α2 and miR-21-β2 is shown in SEQ ID No.13.

[0023] A kit for detecting different types of targets comprises the above-mentioned DNA multi-channel-based detection composition and a test strip; the different types of targets include hepatitis B virus (HBV), microRNA-21 (miR-21), adenosine-5'-triphosphate (ATP), thrombin, and terminal deoxynucleotidyl transferase (TdT).

[0024] According to a preferred embodiment of the present invention, the test strip includes a base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad sequentially connected and attached to the base plate; the nitrocellulose membrane is provided with a detection line and a quality control line.

[0025] Preferably, according to the present invention, DNA-modified AuNPs are immobilized on the conjugate pad, SA-biotin-T-capture strand is immobilized on the detection line, and SA-biotin-C-capture strand is immobilized on the quality control line.

[0026] A method for detecting different types of targets using the above kit comprises the following steps:

[0027] The sample to be tested, molecular beacon, and auxiliary chain are added to the buffer solution and mixed evenly to obtain a reaction system. The reaction system is then incubated at 25°C or 37°C for 30 minutes to obtain a test reaction solution. Finally, the test reaction solution is added dropwise to the sample pad of the test strip, and the test strip color development result is obtained within 15 minutes.

[0028] When the corresponding target exists in the sample to be tested, both the test line and the quality control line will display red; when the corresponding target does not exist in the sample to be tested, the test line will not display color and the quality control line will display red.

[0029] According to the present invention, when the target contained in the sample to be tested is HBV, the auxiliary chain is a combination of HBV-α1 and HBV-β1; in the reaction system, the concentrations of HBV-α1 and HBV-β1 are both 100 nM, and the concentration of the molecular beacon (MB) is 120 nM; the buffer composition is: 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4; the reaction parameters are: incubation at 25°C for 30 min;

[0030] When the target contained in the test sample is miR-21, the auxiliary chain is a combination of miR-21-α2 and miR-21-β2. In the reaction system, the concentrations of miR-21-α2 and miR-21-β2 are both 100 nM, and the concentration of the molecular beacon (MB) is 100 nM. The buffer composition is: 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4. The reaction parameters are: incubation at 25°C for 30 min.

[0031] When the target contained in the test sample is ATP, the auxiliary chain is a combination of ATP-α3 and ATP-β3. In the reaction system, the concentrations of ATP-α3 and ATP-β3 are both 100 nM, and the concentration of the molecular beacon (MB) is 100 nM. The buffer composition is: 20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1×SSC, pH = 7.6. The reaction parameters are: incubation at 37°C for 30 min.

[0032] When the target contained in the test sample is thrombin, the auxiliary chain is a combination of thrombin-α4 and thrombin-β4; the reaction system contains: 20 nM concentrations of thrombin-α4, 60 nM concentrations of thrombin-β4, and 60 nM concentrations of molecular beacon (MB); the buffer composition is: 1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4; and the reaction parameters are: incubation at 25°C for 30 min.

[0033] According to the present invention, when the target contained in the test sample is terminal deoxynucleotidyl transferase (TdT), the auxiliary chain is a combination of TdT-α5 and TdT-β5. The specific process is as follows: first, the test sample, dATP, and TdT-α5 are added to a buffer to construct a first reaction system; the concentration of TdT-α5 in the first reaction system is 2.5 μM, the concentration of dATP is 1 mM, and the buffer composition is: 1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4; after incubating the first reaction system at 37°C for 30 minutes, the enzyme is inactivated by heating at 75°C for 10 minutes to obtain an extension product;

[0034] The extension product, TdT-β5, and molecular beacon (MB) were then added to TdT detection buffer to construct a second reaction system. In the second reaction system, the concentrations of the extension product, TdT-β5, and molecular beacon (MB) were 50 nM, 40 nM, and 40 nM, respectively. The TdT detection buffer consisted of 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, and 10 mM KCl, pH 7.4. The second reaction system was incubated at 37°C for 30 min.

[0035] The technical principle of the present invention is as follows:

[0036] like Figure 1 As shown, the present invention designs two auxiliary chains (referred to as α and β) and a molecular beacon (MB). Although α and β each have sequences complementary to the MB, these three chains cannot form a triple complex. When a target is present, it can bind to α and β, respectively, forming a stable α / β triple complex. In this triple complex, α and β sequences complementary to the MB sequences are in close proximity, allowing them to hybridize cooperatively with the MB and open it.

[0037] When the target to be detected is a DNA or RNA nucleic acid sequence, the target sequence hybridizes with two auxiliary strands and then forms a DNA four-way junction with the MB.

[0038] Then, the target to be detected can also be a small molecule that binds to the split aptamer designed on the auxiliary chain, or a protein (the protein has sites that bind to two different aptamer sequences on the auxiliary chain), and then forms a DNA three-way junction with the MB.

[0039] Furthermore, the target to be detected can also be an enzyme, such as an enzyme with DNA chain extension activity. Under the action of the enzyme, the extended sequence on one auxiliary chain hybridizes with the designed complementary sequence on the other auxiliary chain, and then binds to the MB to form a DNA three-way junction.

[0040] The formed multi-link will leave two single-stranded overhangs on the opposite side, which serve as a bridging structure to connect the DNA-modified AuNPs and the capture chains pre-sprayed in the test area of ​​the test paper, and the captured AuNPs will accumulate and color in the detection area.

[0041] The beneficial effects of the present invention are as follows:

[0042] 1. The present invention provides a detection composition based on DNA multi-linkages. The auxiliary chains used (the combination of HBV-α1 and HBV-β1, the combination of miR-21-α2 and miR-21-β2, the combination of ATP-α3 and ATP-β3, the combination of Thrombin-α4 and Thrombin-β4, and the combination of TdT-α5 and TdT-β5) are all designed according to the targets to be detected (HBV, miR-21, ATP, Thrombin and TdT), have the advantages of high sensitivity and specificity, and can effectively detect different types of targets.

[0043] 2. The present invention provides a test kit that can detect different types of targets. When the target is present, whether it is DNA / RNA nucleic acid, small molecule, protein or enzyme, the α auxiliary chain and the β auxiliary chain can be connected together, and further hybridized with MB to form a DNA multi-way node (DNA four-way node or three-way node). The DNA multi-way node further acts as a bridging complex to connect the DNA-modified AuNPs and the capture chain on the test strip detection line, so that the test line is colored, thereby conveniently and intuitively determining the target. Since the test strip does not need to be remade when the target changes, it is only necessary to reasonably change the unlabeled α auxiliary chain and β auxiliary chain. Therefore, the re-modification of nanoparticles, the re-pretreatment of pads / membranes and the assembly and cutting of test paper are avoided, saving a lot of manpower, material and financial costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the detection principle of the present invention based on the DNA multi-node detection composition.

[0045] Figure 2 Transmission electron microscopy image of AuNPs (A) and UV-visible absorption spectra of AuNPs (red) and DNA-modified AuNPs (black) (B).

[0046] Figure 3 Detection of HBV results using a DNA multi-node based detection composition;

[0047] In the figure, A is a schematic diagram of the structure of the HBV-targeting DNA four-way junction;

[0048] B is the polyacrylamide gel electrophoresis characterization of the HBV-targeted DNA quartet; wherein, lane 1: DNA marker; lane 2: HBV; lane 3: α1; lane 4: β1; lane 5: MB; lane 6: Au-probe; lane 7: T-capture strand; lane 8: Au-probe + MB; lane 9: α1 + β1 + MB; lane 10: HBV + α1 + β1 + MB; lane 11: α1 + β1 + MB + Au-probe + T-capture strand; lane 12: HBV + α1 + β1 + MB + Au-probe + T-capture; lane 13: HBV + α1 + MB; lane 14: HBV + α1 + MB + Au-probe + T-capture; lane 15: HBV + β1 + MB; lane 16: HBV + β1 + MB + Au-probe + T-capture;

[0049] C is the visualization result of different concentrations of HBV on the test paper;

[0050] D is the intensity ratio of different HBV concentrations (T / C);

[0051] E is the specific detection result of HBV sequence;

[0052] F is the comparison of the detection performance of different concentrations of HBV in cell lysate (right) and buffer (left).

[0053] Figure 4 Optimization of incubation temperature for HBV detection using DNA multi-channel detection compositions;

[0054] In the figure, A is the visualization result of the test paper without HBV (left) or with HBV (right) at different incubation temperatures; B is the intensity ratio (T / C) without HBV (black) or with HBV (red) at different incubation temperatures.

[0055] Figure 5 Optimizing the incubation time for HBV detection using a DNA multi-channel detection composition;

[0056] In the figure, A is the visualization result of the test paper at different incubation times, without HBV (left) or with HBV (right); B is the intensity ratio (T / C) of the test paper at different incubation times, without HBV (black) or with HBV (red).

[0057] Figure 6 Optimization of buffers for HBV detection using DNA multi-channel detection compositions;

[0058] In the figure, A is the 2+Visualization results of the test paper in the presence of HBV (left) or HBV (right) in buffer solutions with different concentrations; B is the visualization results of the test paper in the presence of HBV (left) or HBV (right) in buffer solutions with different concentrations of Mg 2+ Intensity ratio (T / C) of the absence (black) or presence (red) of HBV in buffer at different concentrations.

[0059] Figure 7 Detection of miR-21 results using a DNA multi-node based detection composition;

[0060] In the figure, A is a schematic diagram of the structure of the miR-21-targeted DNA four-way junction; B is the visualization result of different concentrations of miR-21 on the test paper; C is the intensity ratio (T / C) of different concentrations of miR-21; D is the sequence-specific detection result of miR-21.

[0061] Figure 8 Detecting ATP results for a DNA multi-node based detection composition;

[0062] In the figure, A is a schematic diagram of the structure of the ATP-targeted DNA three-way junction; B is the visualization results on the test paper under different auxiliary chains; C is the intensity ratio (T / C) under different auxiliary chains; D is the visualization results on the test paper at different ATP concentrations; E is the intensity ratio (T / C) of different ATP concentrations; F and G are the specific detection results of ATP; H shows the application of the test paper in the detection of endogenous ATP in lemon juice.

[0063] Figure 9 The results of the DNA multi-channel detection composition for detecting thrombin and TdT;

[0064] In the figure, A is a schematic diagram of the thrombin-targeted DNA three-way junction structure; B is the visualization result of different concentrations of thrombin on the test paper; C is the specific detection result of thrombin; D is a comparison of the detection performance of different concentrations of thrombin in serum matrix (right) and buffer (left); E is a schematic diagram of the TdT-targeted DNA three-way junction structure; F is the visualization result of different concentrations of TdT on the test paper; G is the specific detection result of TdT; H is a comparison of the detection performance of different concentrations of TdT in HeLa cell lysate matrix (right) and buffer (left).

[0065] Figure 10 Intensity ratio for detecting thrombin by a DNA multi-node based detection composition;

[0066] In the figure, A is the intensity ratio of different concentrations of thrombin; B is the intensity ratio of specific detection of thrombin; C is the intensity ratio of different concentrations of thrombin in serum matrix (right) and buffer (left).

[0067] Figure 11 Intensity ratio for detecting TdT by a DNA multi-junction based detection composition;

[0068] In the figure, A is the intensity ratio of different concentrations of TdT; B is the intensity ratio of specific detection of TdT; C is the intensity ratio of different concentrations of TdT in HeLa cell lysate matrix (right) and buffer (left). DETAILED DESCRIPTION

[0069] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0070] Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the claims of the present invention.

[0071] Reagents and materials:

[0072] DNA sequences and RNA sequences (Table 1) were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0073] Chloroauric acid trihydrate (HAuCl4·3H2O), sodium citrate (Na3C6H5O7·2H2O), tris(2-carboxyethyl)phosphine (TCEP), adenosine 5′-triphosphate disodium salt hydrate (ATP), thrombin (from bovine plasma), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA).

[0074] Terminal deoxynucleotidyl transferase (TdT), TdT reaction buffer (5×), and SYBR Gold nucleic acid stain were provided by Thermo Scientific.

[0075] Ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), 40% (w / v) acrylamide / bisacrylamide solution (19:1), tris(hydroxymethylaminomethane), streptavidin, GTP solution, UTP solution, TTP solution, CTP solution, deoxyadenosine triphosphate (dATP), T4 DNA ligase, and α-amylase were provided by Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0076] ATP solution was provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China).

[0077] Fetal bovine serum was provided by Shanghai Xiaopeng Biotechnology Co., Ltd. (Shanghai, China). Purinic / pyrimidinic endonuclease-1 (APE1) was purchased from New England Biolabs (Beverly, MA).

[0078] All other reagents were of analytical grade. Sample pads, conjugate pads, nitrocellulose membranes, and absorbent pads were provided by Shanghai Jinbiao Biotechnology Co., Ltd. (Shanghai, China).

[0079] UV-visible absorption spectra were recorded by TU-1901 spectrometer (Persee, China). Transmission electron microscopy (TEM) measurements were performed by transmission electron microscope (JSM-6700F) at an accelerating voltage of 200 kV (JEOL, Japan). Polyacrylamide gels were prepared by Gel Doc™ XR + Imaging was performed using an imaging system (Bio-RAD Laboratories, Inc., USA). Gold spraying and film streaking were performed using an HM3035XYZ 3D gold streaking instrument (Shanghai Jinbiao Biotechnology Co., Ltd., China). Test strips were cut using a ZQ2002 programmable strip cutter (Shanghai Jinbiao Technology Co., Ltd., China).

[0080] Table 1. Oligonucleotide sequences used in the examples

[0081]

[0082]

[0083] In the table: The complementary bases between the MB and the α auxiliary strand are single underlined. The complementary bases between the MB and the β auxiliary strand are double underlined. The portion where the MB binds to the Au probe and the portion where the Au probe binds to the C capture strand are italicized and single underlined. The portion where β1-β5 interacts with the T capture strand is wavy underlined. Mismatched bases in HBV and miR-21 are bolded. Supplementary thymidine bases are italicized.

[0084] Example 1

[0085] 1. The preparation method of DNA-modified AuNPs is as follows:

[0086] Prepare 10 mL of 38.8 mM sodium citrate aqueous solution and 50 mL of 10 mM HAuCl4 aqueous solution; heat 45 mL of ultrapure water to boiling, add 5 mL of 10 mM HAuCl4 aqueous solution under vigorous stirring, and after 2 minutes, quickly add 5 mL of sodium citrate aqueous solution to the HAuCl4 aqueous solution while vigorously stirring. React for 10 minutes under heating and boiling state. After the reaction is completed, stir and cool to 25°C, and filter through a 0.22 μm filter to obtain 13 nm gold nanoparticles (AuNPs).

[0087] Thiolated DNA (Au-probe) was incubated with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at a molar ratio of 1:50 at 25°C for 2 hours to reduce disulfide bonds and obtain TCEP-treated DNA.

[0088] TCEP-treated DNA and AuNPs were mixed at a molar ratio of 100:1 and incubated at 25°C for 16 hours to obtain a mixture. Then, 10 μL of saline buffer (55 mM Tris-HCl, 2.2 M NaCl, pH 8.0) was added to the mixture every 40 minutes to a final concentration of 5 mM Tris-HCl and 200 mM NaCl, resulting in a saline solution. The saline solution was then gently stirred at room temperature for 24 hours. The solution was then transferred to a centrifuge tube and centrifuged three times at 14,000 rpm for 30 minutes at 4°C. The solution was then washed with wash buffer (10 mM Tris-HCl, pH 8.0) to separate the DNA-modified AuNPs from excess DNA. The resulting product was resuspended in 100 μL of wash buffer to obtain DNA-modified AuNPs (DNA-AuNPs) and stored at 4°C in the dark.

[0089] 2. A drop of the AuNPs solution prepared in this example was added to the carbon-coated copper grid and dried at 25°C to prepare a sample for TEM characterization. Characterization was performed on a JSM-6700F transmission electron microscope with an accelerating voltage of 200 kV. The results are shown in FIG. Figure 2 As shown in A.

[0090] The UV-visible absorption spectra of the AuNPs and DNA-modified AuNPs prepared in this example were measured. Figure 2 As shown in B.

[0091] Depend on Figure 2 As shown in Figures A and B, the AuNPs and DNA-modified AuNPs in this example were successfully synthesized.

[0092] 3. The present invention detects five different types of targets: hepatitis B virus (HBV), microRNA-21 (miR-21), adenosine 5'-triphosphate (ATP), thrombin, or terminal deoxynucleotidyl transferase (TdT). The auxiliary chains are a combination of HBV-α1 and HBV-β1, and / or a combination of miR-21-α2 and miR-21-β2, and / or a combination of ATP-α3 and ATP-β3, and / or a combination of thrombin-α4 and thrombin-β4, and a combination of TdT-α5 and TdT-β5, corresponding to the five targets.

[0093] Among them, HBV-α1 and HBV-β1 are designed based on the HBV gene segment shown in SEQ ID No. 12, and miR-21-α2 and miR-21-β2 are designed based on miR-21 shown in SEQ ID No. 13.

[0094] 4. Preparation of test strips

[0095] The sample pad was immersed in a glass fiber solution (20 mM Tris-HCl, 150 mM NaCl, 0.25% Triton X-100, 1.5% PVP, pH 8.0) for 5 min and then dried at 37°C for 4 hours.

[0096] A solution containing 10% sucrose and 50 nM DNA-modified AuNPs was evenly sprayed on the conjugate pad at a flow rate of 3 μL / cm and dried at 37° C. for 4 hours.

[0097] Streptavidin (SA, 2 mg / mL) and 100 μM biotin-labeled DNA (T-capture strand, C-capture strand) were mixed in a 1:1 volume ratio and incubated at room temperature for 1 hour. The mixture was then transferred to a 30 kDa ultrafiltration centrifuge tube and centrifuged twice (14,000 rpm, 30 min). After washing with PBS buffer, SA-biotin-DNA complexes, SA-biotin-T-capture strand and SA-biotin-C-capture strand, were obtained. The SA-biotin-DNA complexes were then evenly sprayed onto the nitrocellulose membrane test line and quality control line at a flow rate of 0.4 μL / cm.

[0098] The treated nitrocellulose membrane, conjugate pad, sample pad and absorbent pad were assembled on the polyester base in sequence. The spacing between the test line and the quality control line was 0.2 cm, and the adjacent layers overlapped by 2 mm to ensure the continuity of the flow. Finally, the assembled test paper was cut into 2 mm wide test strips and stored in a dry and dark place.

[0099] Example 2

[0100] 1. DNA characterization by four-way polypropylene gel electrophoresis is as follows:

[0101] Au-probe, T-capture strand, HBV (SEQ ID No. 12), molecular beacon (MB), HBV-α1 and HBV-β1 were grouped as follows:

[0102] ①, Au-probe+MB;

[0103] ②, α1+β1+MB;

[0104] ③, HBV+α1+β1+MB;

[0105] ④, α1+β1+MB+Au-probe+T-capture strand;

[0106] ⑤, HBV+α1+β1+MB+Au-probe+T-capture;

[0107] ⑥, HBV+α1+MB;

[0108] ⑦, HBV+α1+MB+Au-probe+T-capture;

[0109] ⑧, HBV+β1+MB;

[0110] ⑨, HBV+β1+MB+Au-probe+T-capture.

[0111] The above 9 groups were then added to a buffer solution (20mM Tris-HCl, 5mM MgCl2, 60mM NaCl, 10mM KCl, pH = 7.4) and mixed evenly to obtain 9 reaction systems; in each reaction system, the concentration of HBV was 150nM, and the concentrations of α1, β1, MB, Au-probe, and T-capture were 300nM; the reaction system was then incubated at 25°C for 30min to obtain 9 reaction solutions containing the HBV-targeting DNA quartet. Among them, the structure of the HBV-targeting DNA quartet is as follows Figure 3 As shown in A.

[0112] Depend on Figure 3 A shows that HBV viral DNA fragments can hybridize with the 3'-terminal domain of α1 and the 5'-terminal domain of β1, and after hybridization, further hybridize with MB through the 5'-terminal domain of α1 and the 3'-terminal domain of β1 to form a DNA four-way node.

[0113] The above 9 groups of reaction solutions were subjected to polyacrylamide gel electrophoresis, and the individual HBV, α1, β1, MB, Au-probe and T-capture strands were used as controls. The results are shown in the figure. Figure 3 As shown in B.

[0114] Depend on Figure 3 B shows that in the presence of HBV DNA sequences, a migration band corresponding to the DNA quartet composed of HBV, α1, β1, and MB can be observed (lane 10), while in the absence of HBV, no DNA quartet can be formed (lane 9). In addition, after adding the DNA sequence to be modified on AuNPs (Au-probe) and fixed on the detection line (T-capturestrand), a band with the slowest migration rate appears, which is the DNA quartet complex of the two sequences hybridized (lane 10), indicating that the connecting complex can play the role of bridging complex.

[0115] 2. Add HBV, MB, α1, and β1 to a buffer solution (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4), mix well, adjust the HBV concentration to a gradient of 0 nM, 5 nM, 10 nM, 15 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, and 130 nM, and keep the concentrations of HBV-α1, HBV-β1, and MB at 100 nM to obtain 10 reaction systems; then incubate each reaction system at 25 ° C for 30 min to obtain 10 reaction solutions to be tested; finally, add the 10 reaction solutions to be tested to the sample pad of the test strip respectively. After 15 min, the color development results are as follows: Figure 3 C, the intensity ratio (T / C) of different HBV concentrations is as follows Figure 3 As shown in D.

[0116] Depend on Figure 3 As shown in Figures C and D, as the HBV DNA concentration increases, the color of the test line deepens, while the color of the control line weakens, indicating that more tetrahedral junctions are formed and captured by the test line. The intensity ratio of the test line to the control line shows a good linear relationship with HBV concentration in the range of 0 to 130 nM. The detection limit calculated by the 3σ method is 0.63 nM. This detection limit meets diagnostic requirements, especially in the early stages of immune tolerance after HBV infection.

[0117] 3. Add HBV, MB, α1, and β1 to a buffer solution (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4), mix well, and obtain an HBV reaction system; in the HBV reaction system, the concentrations of α1, β1, and MB are all 100 nM, and the concentration of HBV is 50 nM.

[0118] Then HBV was replaced with 1-base mismatched HBV, 2-base mismatched HBV, and 3-base mismatched HBV to obtain three groups of mutation reaction systems.

[0119] The HBV reaction system, three groups of mutant reaction systems and blank reaction system (Blank, only buffer) were incubated at 25 ° C for 30 minutes to obtain 5 groups of test reaction solutions; finally, the 5 groups of test reaction solutions were added dropwise to the sample pad of the test strip. After 15 minutes, the color development results and the intensity ratio (T / C) results of different HBV sequences were as follows: Figure 3 As shown in E.

[0120] Depend on Figure 3E It can be seen that the detection composition and test paper of the present invention have extremely high specificity for HBV sequences and excellent discrimination ability for base mutations. Even a single base mismatch will not cause the detection line to be colored equivalent to the background.

[0121] 4. HBV was added to HeLa cell lysate (10%, V / V) and buffer (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) to obtain biomatrix solutions and buffer solutions with final HBV concentrations of 0, 30, 50, and 80 nM.

[0122] MB, α1, and β1 were then added to the biological matrix solution and buffer solution to construct a reaction system. Each reaction system was then incubated at 25°C for 30 minutes to obtain the reaction solution to be tested. Finally, the reaction solution to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results and the intensity ratio (T / C) of different HBV sequences were obtained. Figure 3 As shown in F.

[0123] Depend on Figure 3 It can be seen that when viral HBV DNA is added to 10% cell lysate, the detection composition and test paper of the present invention can still maintain their detection effectiveness, which is mainly due to the high stability of the DNA multi-link.

[0124] Example 3

[0125] 1. Add HBV, MB, α1, and β1 to a buffer solution (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) and mix well to obtain a reaction system. A reaction system without HBV was used as a control. In the HBV reaction system, the concentrations of α1, β1, and MB were all 100 nM, and the concentration of HBV was 50 nM.

[0126] Then the reaction system was incubated at 10, 25, and 37 ° C for 30 minutes to obtain the reaction solution to be tested; finally, the reaction solution to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development result was as follows: Figure 4 As shown in A, the intensity ratio (T / C) of the presence and absence of HBV is as follows Figure 4 As shown in B.

[0127] Depend on Figure 4 It is clear that temperature affects DNA hybridization dynamics and the stability of the through-link structure. When the temperature is too low, MB tends to exist in a more stable secondary structure, which is not conducive to opening. When the temperature is too high, the through-link structure is not stable and the binding force is weakened. The results show that 25°C is the optimal reaction temperature.

[0128] 2. Add HBV, MB, α1, and β1 to a buffer solution (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) and mix well to obtain a reaction system. A reaction system without HBV was used as a control. In the HBV reaction system, the concentrations of α1, β1, and MB were all 100 nM, and the concentration of HBV was 50 nM.

[0129] Then the reaction system was incubated at 25°C for 10, 20, 30, and 40 minutes respectively to obtain the reaction solution to be tested; finally, the reaction solution to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development result was as follows: Figure 5 As shown in A, the intensity ratio (T / C) of the presence and absence of HBV is as follows Figure 5 As shown in B.

[0130] Depend on Figure 5 As can be seen, as the reaction time increases, the reaction degree increases, the T line color gradually deepens, and the reaction approaches saturation at 30 minutes; the background value remains basically stable. Therefore, 30 minutes is selected as the optimal reaction time.

[0131] 3. Add HBV, MB, α1, and β1 to different Mg 2+ The reaction system was prepared by mixing the mixture in a buffer solution of 100 μg / mL and mixing evenly to obtain a reaction system. A reaction system without HBV was used as a control. In the HBV reaction system, the concentrations of α1, β1, and MB were all 100 nM, and the concentration of HBV was 50 nM.

[0132] Then the reaction system was incubated at 25°C for 30 minutes to obtain the reaction solution to be tested; finally, the reaction solution to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development result was as follows: Figure 6 As shown in A, the intensity ratio (T / C) of the presence and absence of HBV is as follows Figure 6 As shown in B.

[0133] Different Mg 2+ The buffer compositions of the concentrations were the same as those in points 1 and 2, except that the MgCl2 concentrations were 1 mM, 5 mM, and 10 mM, respectively.

[0134] Depend on Figure 6 It can be seen that when Mg 2+ When the concentration is 1mM, the formation tendency of the node is weak and the color of the test paper is light; when the concentration of Mg is 1mM, the formation tendency of the node is weak and the color of the test paper is light. 2+ When the concentration was 5mM, the background was basically unchanged compared to 1mM, and the color development was significantly enhanced; 2+ At a concentration of 10 mM, the system's ability to form complex multiplexes increased, the background increased, and the T-line color did not significantly deepen. Therefore, a magnesium ion concentration of 5 mM was selected for subsequent experiments.

[0135] Example 4

[0136] 1. Add miR-21, MB, miR-21-α2 and miR-21-β2 to buffer (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) and mix well to obtain a reaction system; in the reaction system, the concentrations of miR-21-α2 and miR-21-β2 were both 100 nM, the concentration of molecular beacon (MB) was 100 nM, and the concentration of miR-21 was a gradient of 0 nM, 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, and 100 nM, for a total of 8 reaction systems.

[0137] Then, each reaction system was incubated at 25°C for 30 min to obtain 8 reaction solutions containing miR-21 targeting DNA quartet. The structure of the miR-21 targeting DNA quartet is as follows: Figure 7 As shown in A.

[0138] Depend on Figure 7 As shown in Figure 3, miR-21 can form a four-way junction with miR-21-α2, miR-21-β2, and MB, which can serve as a link to connect DNA-modified AuNPs to the capture strand of the test line.

[0139] Finally, each group of test reaction solution was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results were as follows: Figure 7 As shown in B, the intensity ratio (T / C) of different concentrations of miR-21 is as follows Figure 7 As shown in C.

[0140] Depend on Figure 7 As shown in Figures B and C, as the concentration of miR-21 increased from 0 nM to 100 nM, the test line on the prepared test paper gradually colored, and the concentration ratio of the test line to the control line was linearly related to the miRNA concentration, with a detection limit of 3.78 nM.

[0141] 2. Add miR-21, MB, miR-21-α2 and miR-21-β2 to buffer (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4), mix well, and obtain a reaction system; in the reaction system: the concentrations of miR-21-α2 and miR-21-β2 are both 100 nM, the concentration of MB is 100 nM, and the concentration of miR-21 is 60 nM.

[0142] Then miR-21 was replaced with 1-base mismatched miR-21, 2-base mismatched miR-21, and 3-base mismatched miR-21 to obtain three groups of mutation reaction systems.

[0143] The miR-21 reaction system, the three mutant reaction systems, and the blank reaction system (Blank, buffer only) were incubated at 25°C for 30 minutes to obtain five groups of test reaction solutions. Finally, the five groups of test reaction solutions were added dropwise to the sample pad of the test strip. After 15 minutes, the color development results and the intensity ratio (T / C) of different HBV sequences were obtained. Figure 7 As shown in D.

[0144] Depend on Figure 7 D shows that the detection line will show color only when the detection sequence is the miR-21 sequence. When one, two or three mismatch base mutations occur, the detection line will not show color, which indicates that the detection composition and test paper of the present invention have extremely high specificity for the miR-21 sequence.

[0145] Example 5

[0146] 1. Group the different auxiliary chains ATP-α3 (α-1), ATP-α1 (α), ATP-α2 (α-2), ATP-β3 (β-2), ATP-β1 (β) and ATP-β2 (β-1) according to the following:

[0147] (1) α+β; (2) α-1+β-1; (3) α-2+β-2; (4) α-1+β-2. ATP, MB, and different auxiliary chain combinations were added to a buffer solution (20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1×SSC, pH=7.6) and mixed well to obtain four reaction systems; in the reaction systems, the concentrations of α, β, α-1, β-1, α-2, and β-2 were all 100 nM, the concentration of the molecular beacon (MB) was 100 nM, and the concentration of ATP was 500 μM.

[0148] Then, each reaction system was incubated at 37°C for 30 min to obtain 4 reaction solutions containing ATP-targeted DNA three-way junctions. The structure of the ATP-targeted DNA three-way junction is as follows: Figure 8 As shown in A.

[0149] Finally, each group of test reaction solution was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results were as follows: Figure 8 As shown in B, the intensity ratio of different auxiliary chains (T / C) is as follows Figure 8 As shown in C.

[0150] Depend on Figure 8As shown in B and C, when the 5'-terminal domain of the auxiliary chain α and the 3'-terminal domain of β each have 10 bases, as in nucleic acid detection, the test line will show color even in the absence of ATP. This may be due to the weak interaction between the two split aptamers, which hybridize with the MB to form a connection complex. In order to reduce the nonspecific formation of the complex, the inventors of the present application shortened the arms that bind to MB in α and β by one or two bases, and named them α-1 / α-2 and β-1 / β-2, respectively. Select the appropriate sequence based on the color depth of the T line and C line of the test paper. α (α-1, i.e., α3) that is shortened by one base and β (β-2, i.e., β3) that is shortened by two bases in the MB binding arm will obtain a low background in the absence of ATP, and a high signal in the presence of ATP, and will be used for subsequent detection.

[0151] 2. Add ATP, MB, ATP-α3 and ATP-β3 to buffer (20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1× SSC, pH = 7.6) and mix well to obtain a reaction system; in the reaction system: the concentrations of ATP-α3 and ATP-β3 are both 100 nM, the concentration of MB is 100 nM, and the gradient concentration of ATP is 0 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, and 700 μM, for a total of 8 reaction systems.

[0152] Then, each reaction system was incubated at 37°C for 30 minutes to obtain 8 groups of reaction solutions containing ATP-targeted DNA three-way junctions; finally, each group of reaction solutions to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results were as follows: Figure 8 As shown in D, the intensity ratio (T / C) of different ATP concentrations is as follows Figure 8 As shown in E.

[0153] Depend on Figure 8 As shown in D and E, as the ATP concentration increases from 0 μM to 700 μM, the color of the detection line gradually deepens ( Figure 8 D), and the intensity ratio is linearly correlated with ATP concentration. As little as 50 μM ATP can cause visible coloration, resulting in a calculated limit of detection of 43 μM. Considering that ATP concentrations in living cells and mitochondria range from 1 to 10 mM, this limit of detection should meet practical detection needs.

[0154] 3. Add ATP, MB, ATP-α3 and ATP-β3 to a buffer solution (20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1×SSC, pH = 7.6), mix well, and obtain a reaction system; in the reaction system, the concentrations of ATP-α3 and ATP-β3 are both 100 nM, the concentration of MB is 100 nM, and the concentration of ATP is 500 μM.

[0155] Then ATP was replaced with UTP, TTP, GTP and CTP to obtain four groups of specific detection reaction systems.

[0156] The ATP reaction system, four groups of specific detection reaction systems and a blank reaction system (Blank, only buffer) were incubated at 37°C for 30 minutes to obtain six groups of test reaction solutions. Finally, the six groups of test reaction solutions were added dropwise to the sample pad of the test strip. After 10 minutes, the color development results and intensity ratio (T / C) results of different targets (ATP, UTP, TTP, GTP and CTP) were obtained, as shown in the following figure: Figure 8 F and 8G.

[0157] Depend on Figure 8 As can be seen from Figures 8F and 8G, the test line only develops color for ATP, but not for its analogs UTP, TTP, GTP or CTP, which indicates that the detection composition and test paper of the present invention have extremely high specificity for ATP.

[0158] 4. Soak a cotton swab in fresh lemon juice, then insert the cotton swab soaked in lemon juice into 200 μL cell lysis buffer and incubate at 4°C for 5 min; the incubated mixture is centrifuged at 8000 rpm for 4 min, and the supernatant is diluted with an equal volume of buffer (20 mM Tris, 10 mM MgCl2, 200 mM NaCl, 1× SSC, pH = 7.6) and used as the test sample.

[0159] The test sample, MB, ATP-α3, and ATP-β3 were added to a buffer solution (20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1×SSC, pH = 7.6) and mixed evenly to obtain a reaction system; in the reaction system, the concentrations of ATP-α3 and ATP-β3 were both 100 nM, and the concentration of MB was 100 nM.

[0160] Then the reaction system was incubated at 37°C for 30 minutes to obtain the reaction solution to be tested; finally, the reaction solution to be tested was added dropwise to the sample pad of the test strip. After 15 minutes, the color development result was as follows: Figure 8 As shown in H.

[0161] Depend on Figure 8As can be seen from FIG, the test line appears red, which indicates that the detection composition and the test paper of the present invention can be used to detect endogenous ATP in fruits.

[0162] Example 6

[0163] 1. Thrombin, MB, thrombin-α4, and thrombin-β4 were added to a buffer solution (1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4) and mixed well to obtain a reaction system; in the reaction system, the concentrations of thrombin-α4, thrombin-β4, and molecular beacon (MB) were 20 nM, 60 nM, and 60 nM, respectively. The thrombin concentrations were in a gradient of 0 U / mL, 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, and 50 U / mL, for a total of 5 reaction systems.

[0164] Then, each reaction system was incubated at 25°C for 30 min to obtain 5 reaction solutions containing thrombin-targeted DNA three-way junctions. The structure of the thrombin-targeted DNA three-way junction is as follows: Figure 9 As shown in A.

[0165] Depend on Figure 9 As shown in Figure 3, in the presence of thrombin, the thrombin aptamers in α4 and β4 can be linked together by recognizing two different sites (heparin and fibrinogen) that bind to thrombin, and then hybridize with MB to form a three-way junction, connecting the DNA-modified AuNPs to the detection line.

[0166] Finally, each group of test reaction solution was added dropwise to the sample pad of the test strip. After 10 minutes, the color development results were as follows: Figure 9 As shown in B, the intensity ratio (T / C) of different concentrations of thrombin is as follows Figure 10 As shown in A.

[0167] Depend on Figure 9 As shown in Figures B and 10A, the color of the test line gradually intensifies as the thrombin concentration increases from 0 U / mL to 50 U / mL, and the intensity ratio (T / C) shows a good linear relationship with the thrombin concentration. The detection limit is 8 U / mL, which meets the requirement for detecting thrombin levels of 50-100 U / mL during coagulation.

[0168] 2. Add thrombin, MB, Thrombin-α4 and Thrombin-β4 to a buffer solution (1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4), mix well, and obtain a reaction system; in the reaction system, the concentration of Thrombin-α4 is 20 nM, the concentration of Thrombin-β4 is 60 nM, the concentration of MB is 60 nM, and the concentration of thrombin is 50 U / mL.

[0169] Then, thrombin was replaced with IgG, BSA, and α-amylase to obtain three groups of specific detection reaction systems.

[0170] The thrombin reaction system, three groups of specific detection reaction systems and blank reaction system (Blank, only buffer) were incubated at 25 ° C for 30 minutes to obtain 5 groups of test reaction solutions; finally, the 5 groups of test reaction solutions were added dropwise to the sample pad of the test strip. After 15 minutes, the color development results and specificity of different targets (IgG, BSA, α-amylase) were as follows: Figure 9 C, as shown in 10B.

[0171] Depend on Figure 9 C and 10B show that the test line on the test paper will appear red only when detecting thrombin, while the test line on the test paper will not appear red when detecting other similar proteins such as IgG, BSA or α-amylase, which is consistent with the quantitative intensity ratio result.

[0172] 3. Add thrombin to serum (10%, V / V) and buffer (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) to obtain a biomatrix solution and a buffer solution with final thrombin concentrations of 0 and 50 U / mL, respectively.

[0173] MB, Thrombin-α4 and Thrombin-β4 were then added to the biological matrix solution and buffer solution to construct a reaction system. Each reaction system was then incubated at 25°C for 30 minutes to obtain the test reaction solution. Finally, the test reaction solution was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results and the intensity ratio (T / C) in different media were as follows: Figure 9 D, as shown in 10C.

[0174] Depend on Figure 9 D and 10C show that the visualization results and intensity ratios of thrombin in buffer and 10% serum are basically consistent, indicating that the reagent strip can be used for the detection of thrombin in serum.

[0175] Example 7

[0176] 1. TdT, dATP, and TdT-α5 were added to a buffer solution (1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4) to construct a first reaction system. In this first reaction system, the concentration of TdT-α5 was 2.5 μM, the concentration of dATP was 1 mM, and the concentration of TdT was a gradient of 0 U / μL, 0.02 U / μL, 0.05 U / μL, 0.1 U / μL, 0.3 U / μL, 0.5 U / μL, and 1 U / μL, for a total of 7 reaction systems. Each first reaction system was incubated at 37°C for 30 min and then heated at 75°C for 10 min to inactivate the enzyme, yielding 7 sets of extension products.

[0177] Then, each group of extension products, TdT-β5 and MB were added to TdT detection buffer (20mM Tris-HCl, 5mMMgCl2, 60mM NaCl, 10mM KCl, pH=7.4) to construct a second reaction system; in the second reaction system, the concentration of the extension product was 50nM, the concentration of TdT-β5 was 40nM, and the concentration of MB was 40nM; each group of second reaction systems was incubated at 37°C for 30min to obtain 7 groups of reaction solutions containing TdT-targeted DNA three-way junctions; among which, the structure of the TdT-targeted DNA three-way junction is as follows Figure 9 As shown in E.

[0178] Depend on Figure 9 F shows that under the action of TdT, the 3'-end of α5 uses dATPs as raw materials to extend adenosine nucleotides, which hybridize with the 20-mer poly T sequence at the 5' end of β5.

[0179] Finally, each group of test reaction solution was added dropwise to the sample pad of the test strip. After 15 minutes, the color development results were as follows: Figure 9 As shown in F, the intensity ratio (T / C) of different concentrations of TdT is as follows Figure 11 As shown in A.

[0180] Depend on Figure 9 As shown in Figures F and 11A, the color of the test line gradually intensified with increasing TdT concentration. The T / C intensity ratio was linearly correlated with TdT concentration, ranging from 0 to 1 U / μL, with a detection limit of 0.02 U / μL.

[0181] 2. Add TdT, dATP, and TdT-α5 to a buffer solution (1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4) to construct a first reaction system; the concentration of TdT-α5 in the first reaction system is 2.5 μM, the concentration of dATP is 1 mM, and the concentration of TdT is 0.5 U / mL, thus obtaining a first reaction system;

[0182] Then TdT was replaced with T4 DNA ligase, telomerase, and APE1 to obtain three sets of specific detection first reaction systems.

[0183] After incubating the first reaction system of each group at 37°C for 30 minutes, the enzyme was inactivated by heating at 75°C for 10 minutes to obtain 4 groups of extension products. Then, each group of extension products, TdT-β5 and MB were added to TdT detection buffer (20mM Tris-HCl, 5mMMgCl2, 60mM NaCl, 10mM KCl, pH = 7.4) to construct a second reaction system. In the second reaction system, the concentration of extension product was 50nM, the concentration of TdT-β5 was 40nM, and the concentration of MB was 40nM. Each group of second reaction systems and a blank reaction system (Blank, buffer only) were incubated at 37°C for 30 minutes to obtain 5 groups of test reaction solutions. Finally, the 5 groups of test reaction solutions were respectively added dropwise to the sample pad of the test strip. After 10 minutes, the color development results of different targets (T4 DNA ligase, telomerase, APE1) were as follows. Figure 9 As shown in G, the intensity of different targets, such as Figure 11 As shown in B.

[0184] Depend on Figure 9 G and 11B show that other functional DNA nucleases, such as telomerase, DNA ligase or APE1, do not induce the detection line to develop color or increase the intensity ratio, which indicates that the detection composition and test paper of the present invention have extremely high specificity for TdT enzyme.

[0185] 3. Add TdT to HeLa cell lysate (10%, V / V) and buffer (20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4) to obtain a biomatrix solution and a buffer solution with a final TdT concentration of 0 and 0.5 U / mL, respectively.

[0186] According to the method in point 1, the biological matrix solution and buffer solution with a final TdT concentration of 0 and 0.5 U / mL were used as the test samples for detection. The color development results and the intensity ratio (T / C) in different media are shown in the following table. Figure 9 H and 11C.

[0187] Depend on Figure 9 H and 11C show that when TdT is added to HeLa cell lysate (10%) with low TdT expression, the test strip can still develop color normally, indicating that the test strip has potential clinical use in evaluating TdT activity.

Claims

1. A detection composition based on DNA multi-junction, characterized in that, including molecular beacons and auxiliary chains; The nucleotide sequence of the molecular beacon (MB) is shown in SEQ ID No. 1; The auxiliary chain is a combination of HBV-α1 and HBV-β1, and / or a combination of miR-21-α2 and miR-21-β2, and / or a combination of ATP-α3 and ATP-β3, and / or a combination of Thrombin-α4 and Thrombin-β4, and / or a combination of TdT-α5 and TdT-β5; The nucleotide sequence of the HBV-α1 is shown in SEQ ID No. 2; The nucleotide sequence of the HBV-β1 is shown in SEQ ID No. 3; The nucleotide sequence of miR-21-α2 is shown in SEQ ID No. 4; The nucleotide sequence of miR-21-β2 is shown in SEQ ID No. 5; The nucleotide sequence of the ATP-α3 is shown in SEQ ID No. 6; The nucleotide sequence of the ATP-β3 is shown in SEQ ID No. 7; The nucleotide sequence of Thrombin-α4 is shown in SEQ ID No. 8; The nucleotide sequence of Thrombin-β4 is shown in SEQ ID No.9; The nucleotide sequence of TdT-α5 is shown in SEQ ID No. 10; The nucleotide sequence of TdT-β5 is shown in SEQ ID No.

11.

2. The detection composition according to claim 1, wherein The targets detected by the detection composition are hepatitis B virus (HBV), miR-21, ATP, thrombin or terminal deoxynucleotidyl transferase (TdT); the target genes for detecting HBV-α1 and HBV-β1 are shown in SEQ ID No. 12, and the target genes for detecting miR-21-α2 and miR-21-β2 are shown in SEQ ID No.

13.

3. A kit for detecting different types of targets, characterized in that: The invention comprises the above-mentioned detection composition and test strip based on DNA multi-channel; the different types of targets include hepatitis B virus (HBV), microRNA-21 (miR-21), adenosine-5'-triphosphate (ATP), thrombin and terminal deoxynucleotidyl transferase (TdT).

4. The kit according to claim 3, wherein The test strip comprises a base plate, and a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially connected and attached to the base plate; a detection line and a quality control line are provided on the nitrocellulose membrane.

5. The kit according to claim 4, wherein The binding pad is immobilized with DNA-modified AuNPs, the detection line is immobilized with SA-biotin-T-capture strand, and the quality control line is immobilized with SA-biotin-C-capture strand.

6. A method for detecting different types of targets using the kit according to claim 3 for non-diagnostic purposes, characterized in that: The steps are as follows: The sample to be tested, molecular beacon, and auxiliary chain are added to the buffer solution and mixed evenly to obtain a reaction system. The reaction system is then incubated at 25°C or 37°C for 30 minutes to obtain a test reaction solution. Finally, the test reaction solution is added dropwise to the sample pad of the test strip, and the test strip color development result is obtained within 15 minutes. When the corresponding target exists in the sample to be tested, both the test line and the quality control line will display red; when the corresponding target does not exist in the sample to be tested, the test line will not display color and the quality control line will display red.

7. The method for detecting different types of targets according to claim 6, wherein: When the target contained in the test sample is HBV, the auxiliary chain is a combination of HBV-α1 and HBV-β1; the reaction system: the concentrations of HBV-α1 and HBV-β1 are both 100 nM, and the concentration of the molecular beacon (MB) is 120 nM; the buffer composition is: 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.4; the reaction parameters are: incubation at 25°C for 30 minutes; When the target contained in the test sample is miR-21, the auxiliary chain is a combination of miR-21-α2 and miR-21-β2. In the reaction system, the concentrations of miR-21-α2 and miR-21-β2 are both 100 nM, and the concentration of the molecular beacon (MB) is 100 nM. The buffer composition is: 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 10 mM KCl, pH = 7.

4. The reaction parameters are: incubation at 25°C for 30 min. When the target contained in the test sample is ATP, the auxiliary chain is a combination of ATP-α3 and ATP-β3. In the reaction system, the concentrations of ATP-α3 and ATP-β3 are both 100 nM, and the concentration of the molecular beacon (MB) is 100 nM. The buffer composition is: 20 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1×SSC, pH = 7.

6. The reaction parameters are: incubation at 37°C for 30 min. When the target contained in the test sample is thrombin, the auxiliary chain is a combination of thrombin-α4 and thrombin-β4; in the reaction system, the concentration of thrombin-α4 is 20nM, the concentration of thrombin-β4 is 60nM, and the concentration of molecular beacon (MB) is 60nM; the buffer composition is: 1.6mM Na2HPO4, 0.4mM KH2PO4, 27mM NaCl, 0.5mM KCl, 0.01% Tween-20, pH = 7.4; the reaction parameters are: incubation at 25°C for 30min.

8. The method for detecting different types of targets according to claim 6, wherein: When the target contained in the test sample is terminal deoxynucleotidyl transferase (TdT), the auxiliary chain is a combination of TdT-α5 and TdT-β5. The specific process is as follows: first, the test sample, dATP, and TdT-α5 are added to a buffer to establish a first reaction system; the concentration of TdT-α5 in the first reaction system is 2.5 μM, the concentration of dATP is 1 mM, and the buffer composition is: 1.6 mM Na2HPO4, 0.4 mM KH2PO4, 27 mM NaCl, 0.5 mM KCl, 0.01% Tween-20, pH = 7.4; the first reaction system is incubated at 37°C for 30 minutes, and then heated at 75°C for 10 minutes to inactivate the enzyme, thereby obtaining an extension product; The extension product, TdT-β5, and molecular beacon (MB) were then added to TdT detection buffer to construct a second reaction system. In the second reaction system, the concentrations of the extension product, TdT-β5, and molecular beacon (MB) were 50 nM, 40 nM, and 40 nM, respectively. The TdT detection buffer consisted of 20 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, and 10 mM KCl, pH 7.

4. The second reaction system was incubated at 37°C for 30 min.

Images