Use of aptamer in addressability detection

Abstract

A method for detecting the addressability of a nucleic acid target, comprising: (a) preparing an aptamer-target unit by means of amplification; (b) preparing a solid support, and one or more types of capture element immobilized on the surface of and / or in the interior of the solid support; (c) a marker capable of specifically recognizing and / or binding to the aptamer-target unit, the aptamer-target unit, and the capture element forming a complex; and (d) detecting a signal.

Description

Application of aptamer in addressable detection

[0001] Cross-reference to Related Applications

[0002] This disclosure claims the benefit of and priority to Chinese Patent Application No. 202410888549.2, filed July 3, 2024, entitled “Application of aptamer in addressable detection,” the entire contents of which, including any sequence listing and drawings, are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates to the field of biotechnology, in particular to an aptamer-target unit and its application in addressable detection of amplification products of nucleic acid targets. BACKGROUND

[0004] Nucleic acid detection, including DNA detection and RNA detection, shows important value in disease diagnosis, treatment monitoring and prognosis evaluation. With more and more nucleic acid markers being discovered, higher requirements are put forward for nucleic acid detection technology, among which multi-target parallel detection and low-cost on-site and rapid detection are the development trend of nucleic acid detection technology. Three key core problems must be solved in nucleic acid detection process: 1. amplification of nucleic acid sequence to be detected; 2. visual labeling of nucleic acid target molecules; 3. detection of nucleic acid target molecules;

[0005] In the aspect of amplification of nucleic acid sequence to be detected, the most commonly used at present is PCR amplification technology. After nearly twenty years of development, various ways of PCR technology have emerged, which has become a routine technology in the laboratory. It is an important means in modern molecular biology research and a sensitive amplification system. However, whether it is fluorescent quantitative PCR or other PCR amplification technology, the amplification process needs to rely on high-end and expensive instrument equipment, and the detection conditions are high. Nucleic acid isothermal amplification is a substitute method for PCR. Its amplification is realized under constant temperature conditions, without the need for precise control of the thermal cycling system, and the equipment used is neither complex nor expensive; at the same time, isothermal amplification can efficiently amplify the nucleic acid sequence to be detected, achieving comparable amplification efficiency to PCR. The commonly used isothermal amplification includes nucleic acid sequence-dependent amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinant polymerase amplification (RPA), exponential amplification reaction (EXPAR), nicking enzyme amplification (NEAR), whole genome amplification (WGA), or linear and cascade amplification methods, combined with T7 and other transcription systems, which can realize effective amplification of DNA and RNA. However, whether it is PCR technology or isothermal amplification technology, there is a problem of non-specific amplification, in addition, the homogeneous detection process after amplification also has the problem of limited number of target detection.

[0006] In the visualization labeling of nucleic acid target molecules, commonly used nucleic acid labeling molecules are nucleic acid non-specific binding probe molecules, such as SYBR Green, EB, TOTO and other first-generation nucleic acid labeling probes. Such molecules have no sequence specificity in nucleic acid labeling and are only suitable for labeling and detection of single purified targets. Although the second-generation TaqMan probe can specifically report the amplification of specific targets, such probes can only be turned on when they are cut and released in solution, and cannot report the position information of the target, so they are only suitable for detection in homogeneous systems, and the maximum number of targets detected in a single tube is not more than six, and the application scenario is relatively limited. Sequence-specific probes also include hybridization probes and molecular beacons, but these probes need to go through nucleic acid solid-state synthesis and strict purification process, resulting in high cost in use.

[0007] In the visualization labeling of immunization, many excellent reporter groups have been developed, such as dyes, fluorescent dyes, cryptic fluorescent dyes, fluorescent microspheres, quantum dots, carbon dots, colored latex spheres, redox molecules, luminescent molecules, enzymes, colloidal particles, radioactive labels, electrochemical functional groups, enzyme plus detectable enzyme substrate, colloidal gold particles, etc. These labels usually need to be labeled by the immune reaction of antibodies and antigens to achieve specific labeling of the target to be measured. However, antibodies are expensive, and different detection samples need to adjust the type of antibody. At the same time, the preparation of antibodies depends on mammals, and the preparation cycle is long, the cost is high, the batch stability is poor, the storage and transportation conditions are high, etc. Secondly, antibodies also have the problem of insufficient specificity, such as cross-reaction, and not all antigens, such as small molecule toxic targets, can produce effective antibodies. In addition, antibodies also have poor compatibility with post-modification, the modification position is difficult to control accurately, and the modification often affects the activity of the antibody.

[0008] With the rapid development of biotechnology, aptamer technology has rapidly emerged and has been well applied in food safety detection, environmental pollution detection and clinical diagnosis methods. Aptamer is a kind of oligonucleotide sequence, which can exhibit similar properties to antibodies, such as high specificity and high affinity binding to targets. At the same time, the following characteristics of aptamer make it an ideal antibody substitute in the field of small molecule toxicity detection: 1. The molecular range of aptamer is wide, which can be a small molecule such as toxin, metal ion, organic dye, antibiotic, peptide, etc. It solves the problem of antibody difficulty in preparing small molecule toxicity targets; 2. The synthesis of aptamer has good repeatability, low cost, only one sixth of the cost of antibody, and good stability; 3. Aptamer has reversible denaturation, which can be synthesized in vitro, stored and transported at room temperature. Denaturation caused by temperature is reversible, which solves the problems of long synthesis time, high cost, poor repeatability and reproducibility of antibodies; 4. Aptamer is easy to functionalize, and biological modification can realize signal generation, affinity capture, covalent crosslinking, nanocarrier modification, interface fixation, etc. It solves the problem of antibody inactivation; most importantly, aptamer has very good specificity, which can effectively distinguish the subtle differences in the structure of target molecules, and can separate substances with similar structures or cross-reactivity, solving the problem of antibody cross-reaction.

[0009] With the further development of aptamer technology, aptamer has been continuously endowed with new functions, such as fluorescent RNA / DNA aptamer. Fluorescent RNA / DNA aptamer can specifically bind to activated fluorescent dye and activate the fluorescence of the dye. Without removing excess activated fluorescent dye, the labeled target of aptamer can be detected qualitatively, quantitatively, and in terms of position and motion state, greatly promoting the development of nucleic acid field. Currently, developed fluorescent RNA includes Spinach, Broccoli, Mango, Pepper, BiRhoBAST, biSiRA, Civias, Okra, etc. Among them, Pepper effectively improves the problems of low correct folding rate, high fluorescent background and low signal-to-noise ratio of previous fluorescent RNA, and realizes high-sensitivity imaging of RNA. Developed fluorescent DNA includes Mango, Lettuce, etc. Importantly, aptamer is also compatible with amplification system, which can be produced together with nucleic acid target amplification without complex in vitro synthesis and purification process. The expressed aptamer can specifically bind to the ligand of the aptamer, which not only greatly reduces the production cost, but also solves the problem of complex process and non-specific binding of target labeling. Therefore, aptamer is particularly suitable as a reporter group or as a linker group for labeling of target molecules and reporter groups.

[0010] In the detection of nucleic acid targets, addressable detection techniques are widely used, such as lateral flow chromatographic test strip technology, dot blot hybridization technology, gene chip technology, microfluidic technology, and suspension chip technology. Such techniques fix capture elements on a solid support, realize effective enrichment of target concentration through specific capture of the capture elements and labeled target molecules, combine the advantages of flow or heterogeneous system flow rate difference, achieve rapid separation and purification, effectively improve the signal-to-noise ratio and sensitivity in the target detection process, and, by combining the position information of the support or encoding the information of the support, realize single or multiple target detection of samples, solving the problems of complex operation and low automation of traditional nucleic acid blot hybridization technology. However, the labeled probes used in the current addressable detection process are expensive and the labeling process is complex, and there are also problems such as limited signal-to-noise ratio, which to some extent limit the further application of the technology.

[0011] In summary, PCR technology, isothermal amplification technology, and addressable detection technology play an important role in nucleic acid detection, but there are bottlenecks that need to be updated before they can be more widely used.

[0012] To solve the bottleneck problems of PCR technology, isothermal amplification technology, and addressable detection technology in the nucleic acid detection process, the first aspect of the present application provides an aptamer-target unit for addressable detection of nucleic acid targets, which comprises one or more aptamers and one or more nucleic acid targets combined with the one or more aptamers.

[0013] In some embodiments, the types of aptamers can be the same or different.

[0014] In some embodiments, the types of nucleic acid targets can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0015] In some embodiments, the number of aptamers can be one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0016] In some specific embodiments, when the number of aptamers is multiple, each aptamer is independently combined with the nucleic acid target.

[0017] In some specific embodiments, different types of aptamers can be one-to-one combined with different types of nucleic acid targets, or the same type of aptamer can be combined with different types of nucleic acid targets.

[0018] In some embodiments, the aptamer and the nucleic acid target are on the same nucleic acid strand.

[0019] In some specific embodiments, the aptamer and the nucleic acid target are directly linked, the aptamer and the nucleic acid target are operably linked by one or more nucleotide sequences, or the aptamer and the nucleic acid target comprise one or more shared nucleotide sequences.

[0020] In some embodiments, the aptamer and the nucleic acid target are not on the same nucleic acid strand.

[0021] In some specific embodiments, the nucleic acid strand on which the aptamer is located and the nucleic acid strand on which the nucleic acid target is located are bound by affinity or covalent bond, or the nucleic acid strand on which the aptamer is located and the nucleic acid strand on which the nucleic acid target is located are bound by hybridization through base complementary pairing.

[0022] In some embodiments, the aptamer is selected from a DNA aptamer or an RNA aptamer.

[0023] In some embodiments, when the aptamer is a DNA aptamer, the aptamer is a non-G-quadruplex.

[0024] In some embodiments, the aptamer comprises at least one modification, which is independently at one or more selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

[0025] In some specific embodiments, the modification is at the phosphate position of the aptamer, and the modification is a modification conferring resistance to nuclease activity.

[0026] In some more specific embodiments, the modification is selected from one or a combination of phosphorothioate linkage, alkyl phosphotriester linkage, aryl phosphotriester linkage, alkyl phosphonate linkage, aryl phosphonate linkage, hydrogen phosphonate linkage, and alkyl phosphoramidate linkage.

[0027] In some specific embodiments, the modification is at the ribose position of the aptamer, and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analogue of a mirror image.

[0028] In some specific embodiments, the modification occurs at a base position of the aptamer and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5'-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

[0029] In some embodiments, the aptamer is selected from one or a combination of Pepper or mutants thereof, Clivias or mutants thereof, Mango or mutants thereof, Spinach or mutants thereof, Broccoli or mutants thereof, BiRhoBAST or mutants thereof, biSiRA or mutants thereof, Riboglow RNA tags or mutants thereof, Chili or mutants thereof, o-Coral or mutants thereof, DIR2s-apt or mutants thereof, 13-2min or mutants thereof, and MGA or mutants thereof, Okra or mutants thereof.

[0030] In some embodiments, the aptamer has a length of 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further preferably 10-200 nt, further more preferably 10-100 nt.

[0031] In some embodiments, the nucleic acid target is from a virus, a bacterium, a fungus, an animal, a plant, or a synthetic construct.

[0032] In some embodiments, the nucleic acid target is obtained from water, soil, blood, whole blood, white blood cells, peripheral blood, mononuclear cells, blood plasma, blood serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate fluid, tracheal aspirate fluid, intranasal aspirate, synovial fluid, joint aspirate fluid, cells, cell extract, fecal matter, tissue, tissue extract, tissue biopsy, or cerebrospinal fluid.

[0033] The second aspect of the present application provides a method for addressable detection of nucleic acid targets, comprising:

[0034] a) preparing aptamer-target units by amplification, wherein the aptamer-target units comprise one or more aptamers and one or more nucleic acid targets bound to the one or more aptamers;

[0035] b) preparing a solid support and one or more capture elements immobilized on the surface and / or inside of the solid support, the capture elements being capable of specifically capturing the aptamer-target units;

[0036] c) a label that can specifically recognize and / or bind to said aptamer-target unit, wherein said label has a detectable signal after binding to said aptamer-target unit; and

[0037] d) detecting said signal.

[0038] In some embodiments, said amplification is a variable temperature amplification or an isothermal amplification.

[0039] In some specific embodiments, said isothermal amplification is selected from one or a combination of nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), exponential amplification reaction (EXPAR), nicking enzyme amplification reaction (NEAR), whole genome amplification (WGA), linear and cascading amplification methods, and RNA transcription.

[0040] In some embodiments, the species of said aptamer can be the same or different.

[0041] In some embodiments, the species of said nucleic acid target can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0042] In some embodiments, the number of said aptamer can be one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0043] In some specific embodiments, when the number of said aptamer is more than one, each aptamer is independently bound to said nucleic acid target.

[0044] In some specific embodiments, different species of aptamer can be bound to different species of nucleic acid target one-to-one, or the same species of aptamer can be bound to different species of nucleic acid target.

[0045] In some embodiments, said aptamer and said nucleic acid target are located on the same nucleic acid strand.

[0046] In some specific embodiments, said aptamer and said nucleic acid target are directly connected, said aptamer and said nucleic acid target are operably linked by one or more nucleotide sequences, or said aptamer and said nucleic acid target comprise one or more shared nucleotide sequences.

[0047] In some embodiments, the aptamer and the nucleic acid target are not on the same nucleic acid strand.

[0048] In some embodiments, the nucleic acid strand on which the aptamer resides and the nucleic acid strand on which the nucleic acid target resides are amplified independently in the same system.

[0049] In some embodiments, the nucleic acid strand on which the aptamer resides and the nucleic acid strand on which the nucleic acid target resides are mixed after amplification in different systems.

[0050] In some specific embodiments, the nucleic acid strand on which the aptamer resides and the nucleic acid strand on which the nucleic acid target resides are bound by affinity or covalent bond, or the nucleic acid strand on which the aptamer resides and the nucleic acid strand on which the nucleic acid target resides are bound by hybridization through base complementary pairing.

[0051] In some embodiments, the aptamer is selected from a DNA aptamer or an RNA aptamer.

[0052] In some specific embodiments, when the aptamer is a DNA aptamer, the aptamer is a non-G-quadruplex.

[0053] In some embodiments, the aptamer comprises at least one modification, which is independently at one or more selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

[0054] In some specific embodiments, the modification is at the phosphate position of the aptamer, and the modification is a modification that confers resistance to nuclease activity.

[0055] In some more specific embodiments, the modification is selected from one or a combination of phosphorothioate linkage, alkyl phosphotriester linkage, aryl phosphotriester linkage, alkyl phosphonate linkage, aryl phosphonate linkage, hydrogen phosphonate linkage, and alkyl phosphoramidate linkage.

[0056] In some specific embodiments, the modification is at the ribose position of the aptamer, and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analog of a mirror image.

[0057] In some specific embodiments, the modification occurs at a base position of the aptamer and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

[0058] In some embodiments, the aptamer is selected from one or a combination of Pepper or mutants thereof, Clivias or mutants thereof, Mango or mutants thereof, Spinach or mutants thereof, Broccoli or mutants thereof, BiRhoBAST or mutants thereof, biSiRA or mutants thereof, Riboglow RNA tags or mutants thereof, Chili or mutants thereof, o-Coral or mutants thereof, DIR2s-apt or mutants thereof, 13-2min or mutants thereof, and MGA or mutants thereof, Okra or mutants thereof.

[0059] In some specific embodiments, the aptamer is a Fluorescent Light-up aptamer.

[0060] In some embodiments, the aptamer has a length of 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further preferably 10-200 nt, further more preferably 10-100 nt.

[0061] In some embodiments, the nucleic acid target is from a virus, a bacterium, a fungus, an animal, a plant, or a synthetic construct.

[0062] In some embodiments, the nucleic acid target is obtained from water, soil, blood, whole blood, white blood cells, peripheral blood, mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, cerebrospinal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate fluid, tracheal aspirate fluid, intranasal aspirate, synovial fluid, joint aspirate fluid, cells, cell extract, fecal matter, tissue, tissue extract, tissue biopsy, or cerebrospinal fluid.

[0063] In some embodiments, the aptamer-target unit forms a covalent or non-covalent complex with the label.

[0064] In some specific embodiments, when the aptamer-target unit forms a non-covalent complex with the label, the binding dissociation constant (Kd) between the aptamer-target unit and the label is about 0.001 nM to about 100 µM, preferably about 0.01 nM to about 1 µM, more preferably about 0.01 nM to about 10 nM.

[0065] In some embodiments, the dissociation rate (t 1 / 2 ) of the complex formed between the aptamer-target unit and the label is greater than or equal to 30 minutes, preferably the dissociation rate (t 1 / 2 ) is greater than or equal to 120 minutes.

[0066] In some embodiments, the label comprises a ligand that specifically recognizes and / or binds to the aptamer-target unit.

[0067] In some embodiments, the label further comprises a reporter group linked to the ligand, and the reporter group is selected from one or a combination of dyes, nanoparticles, microspheres, redox molecules, luminescent molecules, radioactive labels, electrochemical functional groups, enzymes, and enzymes plus detectable enzyme substrates.

[0068] In some specific embodiments, the dyes are selected from one or a combination of phosphorescent dyes, time-resolved fluorescent dyes, fluorescent dyes, and cryptic fluorescent dyes.

[0069] In some specific embodiments, the microspheres are selected from one or a combination of fluorescent microspheres, quantum dots, carbon dots, colored latex spheres, fluorescent latex spheres, colloidal particles, colloidal gold particles, and oxide clusters.

[0070] In some specific embodiments, the enzyme in the enzymes plus detectable enzyme substrates is horseradish peroxidase (HRP), luciferase (NanoLuc), alkaline phosphatase (AP), urease, or beta-galactosidase.

[0071] In some embodiments, the detectable signal is selected from one or a combination of color signals, light signals, optoacoustic signals, electro-optical signals, electrical signals, magnetic signals, and radioactive signals.

[0072] In some specific embodiments, the light signals are selected from one or a combination of fluorescence, phosphorescence, chemiluminescence, bioluminescence, color, circular dichroism, and Raman spectroscopy.

[0073] In some embodiments, the label has a detectable signal of different intensity before binding to the aptamer-target unit and after binding to the aptamer-target unit.

[0074] In some specific embodiments, when the detectable signal is a fluorescent signal, the fluorescent signal intensity of the label after binding to the aptamer-target unit is enhanced more than 5 times compared to the fluorescent signal intensity of the label before binding to the aptamer-target unit, preferably the fluorescent signal intensity is enhanced more than 10 times or the excitation wavelength shift is more than 10 nm or the emission wavelength shift is more than 10 nm, more preferably the fluorescent signal intensity is enhanced more than 20 times or the excitation wavelength shift is more than 20 nm or the emission wavelength shift is more than 20 nm, further preferably the fluorescent signal intensity is enhanced more than 50 times or the excitation wavelength shift is more than 50 nm or the emission wavelength shift is more than 50 nm.

[0075] In some specific embodiments, the complex formed by the label and the aptamer-target unit is selected from one or a combination of Pepper485, Pepper497, Pepper508, Pepper514, Pepper525, Pepper530, Pepper599, Pepper620, Clivia580, Clivia577, Clivia581, Clivia582, Clivia590, Clivia600, Clivia624, Clivia565, Clivia570, Clivia571, Clivia574, Clivia578, Clivia595, Clivia618, Chili-DMHBI-Imi and Chili-DMHBO.

[0076] In some more specific embodiments, when the aptamer is Pepper or a mutant thereof, the label is a HBC class dye or a derivative thereof.

[0077] In some more specific embodiments, when the aptamer is Mango or a mutant thereof, the label is a hemicyanine class dye, preferably the label is TO1-Biotin or a mutant thereof.

[0078] In some more specific embodiments, when the aptamer is Spinach or a mutant thereof, Broccoli or a mutant thereof, the label is a green fluorescent protein chromophore class dye, preferably the label is DFHBI.

[0079] In some more specific embodiments, when the aptamer is RhoBAST or a mutant thereof, BiRhoBAST or a mutant thereof, the label is a tetramethylrhodamine class derivative, preferably the label is TMR.

[0080] In some more specific embodiments, when the aptamer is biSiRA or a mutant thereof, the label is a SiR class derivative.

[0081] In some more specific embodiments, when the aptamer is a Riboglow RNA tag or a mutant thereof, the label is a Cbl-F class derivative.

[0082] In some embodiments, the complex is formed when the aptamer-target unit binds to the label and then binds to the capture agent.

[0083] In some embodiments, the complex is formed when the aptamer-target unit binds to the capture agent and then binds to the label.

[0084] In some embodiments, the complex is formed when the aptamer-target unit binds to the label and the capture agent simultaneously.

[0085] In some embodiments, the solid support is selected from one or a combination of a polymer bead, an agarose bead, a polystyrene bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a controlled porosity bead, a microtiter bead, a cyclic olefin copolymer bead, a biofilm, a filter paper, a plastic substrate, nylon, a Langmuir-Blodgett film, glass, a germanium substrate, a silicon substrate, a silicon wafer chip, ceramic, a laminate, a flow-through chip, a microsphere, a nanoparticle, a polytetrafluoroethylene substrate, a polystyrene plate, a gallium arsenide substrate, and a gold, silver plate; preferably one or a combination of glass, a silicon substrate, a nitrocellulose membrane, a cellulose acetate membrane, a flow-through chip, and a gold plate; more preferably one or a combination of a nitrocellulose membrane, a cellulose acetate membrane, a nylon membrane, a flow-through chip, glass, a silicon substrate, a silicon wafer chip, a microsphere, and plastic.

[0086] In some embodiments, the capture agent is selected from one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, a morpholino backbone nucleic acid, a peptide nucleic acid, an antigen, an antibody, a hapten, an enzyme, a nanobody, an aptamer, a monosaccharide, a polysaccharide, an affibody, an antibody mimetic, a cellular receptor, a ligand, a lipid, biotin, avidin, streptavidin, exavidin, neutravidin, Traptavidin, a metal, and histidine; preferably one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, an antigen, an antibody, an aptamer, or biotin; more preferably one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, or biotin.

[0087] In some specific embodiments, when the capture agent is a nucleic acid sequence, the nucleic acid sequence comprises at least one modification, and the modification is independently at one or more selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

[0088] In some more specific embodiments, the modification is at the phosphate position of the aptamer, and the modification is a modification that confers resistance to nuclease activity.

[0089] In some more specific embodiments, the modification is selected from one or a combination of phosphorothioate linkage, alkyl phosphotriester linkage, aryl phosphotriester linkage, alkyl phosphonate linkage, aryl phosphonate linkage, hydrogen phosphonate linkage, and alkyl phosphoramidate linkage.

[0090] In some specific embodiments, the modification occurs at a ribose position of the aptamer and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analogue of a mirror body.

[0091] In some specific embodiments, the modification occurs at a base position of the aptamer and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

[0092] In some embodiments, the capture element specifically captures the aptamer-target unit through affinity capture or covalent reaction.

[0093] In some embodiments, the capture element specifically captures the aptamer-target unit directly.

[0094] In some embodiments, the capture element specifically captures the aptamer-target unit through hybridization by base complementarity, capture of antibody with antigen / hapten, capture of chemical tag with substrate, capture of enzyme with substrate, capture of nanoscale enzyme with substrate.

[0095] In some embodiments, the capture element specifically captures the aptamer-target unit through a mediator probe.

[0096] In some embodiments, the mediator probe comprises a probe region and a capture binding region; wherein the probe region has affinity for the aptamer-target unit and the capture binding region has recognition binding affinity for the capture element, the probe region and the capture binding region are independently selected from one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cellular receptor, ligand, lipid, biotin, avidin, streptavidin, neutravidin, Traptavidin, metal, and histidine, preferably one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, antigen, antibody, aptamer, and biotin; more preferably one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, and biotin.

[0097] In some embodiments, the mediator probe further comprises an intermediate linking region; wherein the intermediate linking region is selected from one or a combination of nucleoside, peptide chain, peptide nucleic acid chain, monosaccharide, polysaccharide, PEG chain, macromolecular polymer, short chain chemical linker, and a linker.

[0098] In some embodiments, when the probe region and / or the capture binding region is a nucleic acid sequence, the nucleic acid sequence comprises at least one modification, and the modification is independently at one or more selected from ribose position, deoxyribose position, phosphate position, and base position.

[0099] In some more specific embodiments, the modification is at the phosphate position of the aptamer, and the modification is a modification that confers resistance to nuclease activity.

[0100] In some more specific embodiments, the modification is selected from one or a combination of phosphorothioate linkage, alkylphosphotriester linkage, arylphosphotriester linkage, alkylphosphonate linkage, arylphosphonate linkage, phosphoramide linkage, and alkylphosphoramidate linkage.

[0101] In some more specific embodiments, the modification is at the ribose position of the aptamer, and the modification is selected from one or a combination of 2’-position sugar modification, 2’-amino (2’-NH2), 2’-fluoro (2’-F), 2’-methoxy (2’-OMe), 2’-ethoxy (2’-OEt), 2’-O-aminopropyl modification, 2’-O-alkyl modification, 2’-O-allyl modification, 2’-O-butyl modification, 1-(4’-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analogue of a mirror body.

[0102] In some more specific embodiments, the modification occurs at a base position of the aptamer, and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

[0103] The third aspect of the present application provides a kit for addressable detection of nucleic acid targets, comprising a primer set for amplification of aptamer-target units, a label, a solid support, a capture element, and optionally an instruction of detection method;

[0104] wherein the primer set comprises primers for amplification of one or more nucleic acid targets and the 5' end of the primers further comprises an aptamer-encoding sequence, or the primer set comprises primers for amplification of one or more nucleic acid targets and primers for amplification of one or more aptamers;

[0105] wherein the aptamer-target units are formed by variable-temperature amplification or isothermal amplification; the label has a detectable signal after binding to the aptamer-target units; and the capture element is immobilized on the surface or inside of the solid support and can specifically capture the aptamer-target units.

[0106] In some embodiments, the aptamers can be of the same species or different species.

[0107] In some embodiments, the nucleic acid targets can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 species.

[0108] In some embodiments, the aptamers can be one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0109] In some specific embodiments, when the aptamers are multiple, each aptamer is independently bound to the nucleic acid target.

[0110] In some specific embodiments, different species of aptamers can be bound to different species of nucleic acid targets one-to-one, or the same species of aptamers can be bound to different species of nucleic acid targets.

[0111] In some embodiments, the aptamers and the nucleic acid targets are located on the same nucleic acid strand after amplification.

[0112] In some embodiments, the aptamer and the nucleic acid target are directly linked, the aptamer and the nucleic acid target are operatively linked by one or more nucleotide sequences, or the aptamer and the nucleic acid target comprise one or more shared nucleotide sequences.

[0113] In some embodiments, the aptamer and the nucleic acid target are not on the same nucleic acid strand after amplification.

[0114] In some embodiments, the aptamer and the nucleic acid target are on the same nucleic acid strand after amplification.

[0115] In some embodiments, the aptamer and the nucleic acid target are on different nucleic acid strands after amplification.

[0116] In some embodiments, the aptamer and the nucleic acid target are on the same nucleic acid strand after amplification.

[0117] In some embodiments, the aptamer is selected from a DNA aptamer or an RNA aptamer.

[0118] In some embodiments, the aptamer is a DNA aptamer, the aptamer is not a G-quadruplex.

[0119] In some embodiments, the aptamer comprises at least one modification, the modification is independently at one or more selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

[0120] In some embodiments, the modification is at a phosphate position of the aptamer, and the modification is a modification that confers resistance to nuclease activity.

[0121] In some embodiments, the modification is selected from one or a combination of a phosphorothioate linkage, an alkyl phosphotriester linkage, an aryl phosphotriester linkage, an alkyl phosphonate linkage, an aryl phosphonate linkage, a hydrogen phosphonate linkage, and an alkyl phosphoramidate linkage.

[0122] In some specific embodiments, the modification occurs at a ribose position of the aptamer and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-furanoribose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analog of a mirror body.

[0123] In some specific embodiments, the modification occurs at a base position of the aptamer and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

[0124] In some embodiments, the aptamer is selected from one or a combination of Pepper or a mutant thereof, Clivias or a mutant thereof, Mango or a mutant thereof, Spinach or a mutant thereof, Broccoli or a mutant thereof, BiRhoBAST or a mutant thereof, biSiRA or a mutant thereof, Riboglow RNA tags or a mutant thereof, Chili or a mutant thereof, o-Coral or a mutant thereof, DIR2s-apt or a mutant thereof, 13-2min or a mutant thereof, and MGA or a mutant thereof.

[0125] In some embodiments, the aptamer has a length of 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further preferably 10-200 nt, further more preferably 10-100 nt.

[0126] In some embodiments, the nucleic acid target is from a virus, a bacterium, a fungus, an animal, a plant, or a synthetic construct.

[0127] In some embodiments, the nucleic acid target is obtained from water, soil, blood, whole blood, white blood cells, peripheral blood, mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate fluid, tracheal aspirate fluid, endoscopic aspirate, synovial fluid, joint aspirate, cells, cell extract, stool, tissue, tissue extract, tissue biopsy, or cerebrospinal fluid.

[0128] In some embodiments, the aptamer-target unit forms a covalent or non-covalent complex with the label.

[0129] In some specific embodiments, when the aptamer-target unit forms a non-covalent complex with the label, the dissociation constant (Kd) between the aptamer-target unit and the label is about 0.001 nM to about 100 μΜ, preferably about 0.01 nM to about 1 μΜ, more preferably about 0.01 nM to about 10 nM.

[0130] In some embodiments, the label comprises a ligand that specifically recognizes and / or binds to the aptamer-target unit.

[0131] In some embodiments, the label further comprises a reporter group connected to the ligand, and the reporter group is selected from one or a combination of dyes, nanoparticles, microspheres, redox molecules, luminescent molecules, radioactive labels, electrochemical functional groups, enzymes, and enzymes plus detectable enzyme substrates.

[0132] In some specific embodiments, the dyes are selected from one or a combination of phosphorescent dyes, time-resolved fluorescent dyes, fluorescent dyes, and cryptic fluorescent dyes.

[0133] In some specific embodiments, the microspheres are selected from one or a combination of fluorescent microspheres, quantum dots, carbon dots, colored latex spheres, fluorescent latex spheres, colloidal particles, colloidal gold particles, and oxide clusters.

[0134] In some specific embodiments, the enzyme in the enzymes plus detectable enzyme substrates is horseradish peroxidase (HRP), luciferase (NanoLuc), alkaline phosphatase (AP), urease, or β-galactosidase.

[0135] In some embodiments, the detectable signal is selected from one or a combination of color signals, light signals, optoacoustic signals, electro-optical signals, electrical signals, magnetic signals, and radioactive signals.

[0136] In some specific embodiments, the light signals are selected from one or a combination of fluorescence, phosphorescence, chemiluminescence, bioluminescence, color, circular dichroism, and Raman spectroscopy.

[0137] In some embodiments, the label has a detectable signal of different intensity before binding to the aptamer-target unit and after binding to the aptamer-target unit.

[0138] In some specific embodiments, when the detectable signal is a fluorescent signal, the fluorescent signal intensity of the label after binding to the aptamer-target unit is enhanced more than 5 times, preferably more than 10 times or the excitation wavelength shift is more than 10 nm or the emission wavelength shift is more than 10 nm, more preferably more than 20 times or the excitation wavelength shift is more than 20 nm or the emission wavelength shift is more than 20 nm, further preferably more than 50 times or the excitation wavelength shift is more than 50 nm or the emission wavelength shift is more than 50 nm, compared to the fluorescent signal intensity of the label before binding to the aptamer-target unit.

[0139] In some specific embodiments, the complex formed by the label and the aptamer-target unit is selected from one or a combination of Pepper 485, Pepper 497, Pepper 508, Pepper 514, Pepper 525, Pepper 530, Pepper 599, Pepper 620, Clivia 580, Clivia 577, Clivia 581, Clivia 582, Clivia 590, Clivia 600, Clivia 624, Clivia 565, Clivia 570, Clivia 571, Clivia 574, Clivia 578, Clivia 595, Clivia 618, Chili-DMHBI-Imi, and Chili-DMHBO.

[0140] In some embodiments, the solid support is selected from one or a combination of polymeric beads, agarose beads, polystyrene beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled porosity beads, microtiter beads, cyclic olefin copolymer beads, biofilm, filter paper, plastic substrates, nylon, Langmuir-Blodgett films, glass, germanium substrates, silicon substrates, silicon wafer chips, ceramic, laminates, flow-through chips, microspheres, nanoparticles, polytetrafluoroethylene substrates, polystyrene plates, gallium arsenide substrates, and gold, silver substrates; preferably one or a combination of glass, silicon substrates, nitrocellulose membranes, cellulose acetate membranes, flow-through chips, and gold plates; more preferably one or a combination of nitrocellulose membranes, cellulose acetate membranes, nylon membranes, flow-through chips, glass, silicon substrates, silicon wafer chips, microspheres, and plastic.

[0141] In some embodiments, the capture agent is selected from one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, a morpholino backbone nucleic acid, a peptide nucleic acid, an antigen, an antibody, a hapten, an enzyme, a nanobody, an aptamer, a monosaccharide, a polysaccharide, an affibody, an antibody mimetic, a cellular receptor, a ligand, a lipid, biotin, avidin, streptavidin, neutravidin, Traptavidin, a metal, and histidine; preferably one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, an antigen, an antibody, an aptamer, or biotin; more preferably one or a combination of a natural nucleic acid sequence, an unnatural nucleic acid sequence, or biotin.

[0142] In some embodiments, when the capture agent is a nucleic acid sequence, the nucleic acid sequence comprises at least one modification, and the modification is independently made at one or more selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

[0143] In some specific embodiments, the modification is made at a phosphate position of the aptamer, and the modification is a modification that confers resistance to nuclease activity.

[0144] In some specific embodiments, the modification is selected from one or a combination of a phosphorothioate linkage, an alkyl phosphorotriester linkage, an aryl phosphorotriester linkage, an alkyl phosphonate linkage, an aryl phosphonate linkage, a hydrogen phosphonate linkage, and an alkyl phosphoramidate linkage.

[0145] In some specific embodiments, the modification is made at a ribose position of the aptamer, and the modification is selected from one or a combination of a 2’-position sugar modification, a 2’-amino (2’-NH2), a 2’-fluoro (2’-F), a 2’-methoxy (2’-OMe), a 2’-ethoxy (2’-OEt), a 2’-O-aminopropyl modification, a 2’-O-alkyl modification, a 2’-O-allyl modification, a 2’-O-butyl modification, a 1-(4’-thio-PD-ribofuranose) modification, a 2-O-4-C modification, a L-DNA, and a nucleic acid analog of a mirror body.

[0146] In some specific embodiments, the modification is made at a base position of the aptamer, and the modification is selected from one or a combination of a 5’-position pyrimidine modification, an 8’-position purine modification, a cytosine exocyclic modification, a substitution of 5’-bromouracil, a substitution of 5’-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, a methylation, a 2’-methoxy ethylene (2’-MOE), a 3’ cap, and a 5’ cap.

[0147] In some embodiments, the kit further comprises a mediator probe, the mediator probe comprising a probe region and a capture binding region;

[0148] wherein said probe region has affinity for said aptamer-target unit, said capture binding region has recognition binding power for said capture element, said probe region and said capture binding region are independently selected from one or a combination of natural nucleic acid sequence, non-natural nucleic acid sequence, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cellular receptor, ligand, lipid, biotin, avidin, streptavidin, neutravidin, Traptavidin, metal, and histidine, preferably one or a combination selected from peptide nucleic acid, natural nucleic acid sequence, non-natural nucleic acid sequence, antigen, antibody, aptamer, and biotin; more preferably one or a combination selected from peptide nucleic acid, natural nucleic acid sequence, non-natural nucleic acid sequence, and biotin.

[0149] In some specific embodiments, said mediator probe further comprises an intermediate linking region; wherein said intermediate linking region is selected from one or a combination of nucleoside, peptide chain, peptide nucleic acid chain, monosaccharide, polysaccharide, PEG chain, macromolecular polymer, short chain chemical linker, and a linker.

[0150] In some specific embodiments, when said probe region and / or said capture binding region is a nucleic acid sequence, said nucleic acid sequence comprises at least one modification, and said modification is independently at one or more selected from ribose position, deoxyribose position, phosphate position, and base position.

[0151] In some specific embodiments, said modification is at the phosphate position of said aptamer, and said modification is a modification conferring resistance to nuclease activity.

[0152] In some specific embodiments, said modification is selected from one or a combination of phosphorothioate linkage, alkylphosphotriester linkage, arylphosphotriester linkage, alkylphosphonate linkage, arylphosphonate linkage, hydrogenophosphonate linkage, and alkylphosphoramidate linkage.

[0153] In some specific embodiments, said modification is at the ribose position of said aptamer, and said modification is selected from one or a combination of 2’-position sugar modification, 2’-amino (2’-NH2), 2’-fluoro (2’-F), 2’-methoxy (2’-OMe), 2’-ethoxy (2’-OEt), 2’-O-aminopropyl modification, 2’-O-alkyl modification, 2’-O-allyl modification, 2’-O-butyl modification, 1-(4’-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and nucleic acid analogs of mirror image.

[0154] In some embodiments, the modification occurs at a base position of the aptamer and the modification is selected from one or a combination of a 5'-position pyrimidine modification, an 8'-position purine modification, a cytosine exocyclic modification, a substitution of 5'-bromouracil, a substitution of 5'-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), a 3' cap, and a 5' cap.

[0155] The fourth aspect of the present application provides use of the aptamer-target unit as described above or the kit as described above in the addressable detection of a nucleic acid target.

[0156] Definitions

[0157] Detection of the target in the amplification product

[0158] Aptamer: a single-stranded nucleic acid molecule, which has one or more base-pairing regions and one or more non-pairing regions, and can specifically recognize / combine with a ligand molecule. In the present application, the aptamer can optionally include a sequence for hybridization in addition to the part that can specifically recognize / combine with the ligand molecule.

[0159] Label: In the present application, the term "label" refers to a ligand that can be specifically recognized / combined with an aptamer, or a complex of a ligand and a reporter group. The label has a small molecule, a microsphere, or a nanoparticle with a detectable signal, and the detectable signal includes a signal that can be detected by means of photonic detection, electronic detection, acoustic detection, electrochemical detection, enzymatic detection, magnetic detection, etc.

[0160] Reporter group: In the present application, the term "reporter group" refers to a group with a detectable signal. The detectable signal includes a signal that can be detected by means of photonic detection, electronic detection, acoustic detection, electrochemical detection, enzymatic detection, magnetic detection, etc.

[0161] Color signal: In the present application, the term "color signal" refers to a color that a reporter has under non-excitation conditions such as natural light, which is recognizable by the naked eye. The color is a color signal under the condition of subtractive color principle.

[0162] Enzyme plus detectable substrate: In the present application, the term "enzyme plus detectable substrate" refers to a substance that can react with an enzyme, and the product after the reaction has a detectable signal. The detectable signal refers to a color signal, an absorption wavelength, and an emission wavelength.

[0163] Mediator probe: In the present application, the term "mediator probe" refers to a probe that can specifically bind to a target gene and an aptamer complex at one end, and can bind to a capture unit on an addressable support at the other end, and optionally has a linking group linking the two parts.

[0164] Target gene: The term "target gene" in the present invention refers to a gene containing a gene sequence to be detected, and optionally, the gene further contains an additional sequence allowing nucleic acid hybridization;

[0165] Target: The term "target", "nucleic acid target" or "target gene", "target sequence" in the present invention refers to a nucleic acid sequence to be detected in an amplification product;

[0166] Complementary: The term "complementary" in the present invention refers to two nucleic acid (natural or unnatural) nucleic acid sequences capable of forming hydrogen bonds between each other according to the principle of base pairing (Waston-Crick principle), thereby forming a duplex.

[0167] Hybridization: The term "hybridization" in the present invention refers to the process in which complementary single-stranded nucleic acid (natural or unnatural) molecules form double-stranded nucleic acids;

[0168] The "conditions allowing nucleic acid hybridization" used in the present invention have the meaning commonly understood by those skilled in the art, and can be determined by conventional methods. For example, two nucleic acid molecules with complementary sequences can hybridize under suitable hybridization conditions, which can involve factors such as temperature, pH value, composition and ionic strength of the hybridization buffer, etc., and can be determined according to the length and GC content of the complementary double-stranded nucleic acid molecules.

[0169] Compared with the prior art, the advantages of the present invention include:

[0170] The present invention discloses a method for amplification sample addressable detection using nucleic acid aptamer and its application. The method of the present invention realizes simple and rapid labeling of the target by using the specific binding of aptamer and ligand, and combines the advantages of addressable detection to realize rapid, sensitive and low-cost detection of single or multiple targets. The present invention has the advantages of rapid labeling, good stability, strong specificity, high sensitivity, wide linear range, and accurate quantification. BRIEF DESCRIPTION OF DRAWINGS

[0171] Figure 1. Schematic diagram of aptamer and target gene on the same nucleic acid sequence. A. The aptamer sequence is directly connected to the target sequence; B. The aptamer sequence is connected to the target gene through several nucleotide sequences; C. The aptamer sequence is connected to the target sequence through a shared nucleotide sequence.

[0172] Figure 2. Schematic diagram of aptamer and target gene not on the same nucleic acid sequence; the sequence where the aptamer is located and the sequence where the target gene is located form a target gene-aptamer complex through affinity or covalent bond.

[0173] Figure 3. Schematic diagram of the label of aptamer; A. The ligand of the aptamer directly serves as a label; B. The complex formed by connecting the ligand of the aptamer with a reporter group serves as a label.

[0174] Figure 4. Schematic diagram of mediator probe and its capture; A. Schematic diagram of mediator probe, including probe region capable of specifically binding to target gene-aptamer complex, capture binding region capable of specifically recognizing capture unit on support, and optional intermediate connection region linking probe region and capture binding region; B. Schematic diagram of capture of mediator probe in addressable labeling, target gene-aptamer complex is labeled on support by probe region and target gene-aptamer complex and capture binding region and capture unit recognition.

[0175] Figure 5. Pepper530 as reporter group for detection of Mycoplasma felis amplification marker. Left panel in Figure 5A is the detection result of negative sample on test strip; right panel in Figure 5A is the detection result of positive sample on test strip; Figure 5B is the detection result of fluorescence signal of test strip in Figure 5A using dry test strip reader.

[0176] Figure 6. HBC620 as reporter group, using mediator probe for detection of Mycoplasma felis amplification marker. Left panel is the detection result of negative sample on test strip; right panel is the detection result of positive sample on test strip.

[0177] Figure 7. Clivias574 as reporter group, using mediator probe for detection of Mycoplasma felis amplification marker. Left panel is the detection result of negative sample on test strip; right panel is the detection result of positive sample on test strip.

[0178] Figure 8. bG aptamer as aptamer, complex of ligand connected with dye as reporter group for addressable detection of target on microfluidic chip.

[0179] Figure 9. Microsphere complex labeled with ligand as reporter group for addressable detection on test strip.

[0180] Figure 10. HRP protein labeled with ligand as reporter group for addressable detection on test strip.

[0181] Figure 11. Chemical luminescent group, acrid ester, labeled with ligand as reporter group for addressable detection on gene chip.

[0182] Figure 12. Pepper599 as reporter group for detection of Mycoplasma felis target in NASBA amplification sample. Left panel in Figure 12A is the detection result of negative sample on test strip; middle and right panels in Figure 12A are the detection result of positive sample on test strip; Figure 12B is the detection result of fluorescence signal of test strip in Figure 12A using dry test strip reader.

[0183] Figure 13. Addressable detection of the ligand-labeled methylene blue as reporter on gold electrode.

[0184] Figure 14. A is the structure of RCA probe; B is the schematic diagram of RCA amplification; C is the detection results of the amplification products of different concentrations of target under the microscope after being captured by gene chip; D is the fluorescence intensity of the amplification products of different concentrations of target after being captured by gene chip.

[0185] Figure 15. Fluorescent DNA as a reporter for the detection of Mycoplasma felis amplification labeling. The left image in Figure 15A is the detection result of a negative sample on the test strip; the right image in Figure 15A is the detection result of a positive sample on the test strip; Figure 15B is the detection result of the fluorescence signal of the test strip in Figure 15A using a dry test strip reader.

[0186] Figure 16. Okra as a reporter for the detection of super-multiplex targets in lateral flow detection. DETAILED DESCRIPTION

[0187] The example embodiments are used to describe the present application, but it should be understood that the present application is not intended to be limited to these embodiments, rather, all alternatives, modifications and equivalents which can be included within the scope of the present application as defined by the claims are intended to be embraced by the present application.

[0188] Example:

[0189] Example 1. Application of Pepper 530 as a reporter in lateral flow detection

[0190] Treatment of sample pad:

[0191] a) Preparation of treatment solution:

[0192] b) Spread the glass fiber flat on the glass plate, and evenly spray the corresponding volume using a graduated cylinder or syringe, and roll flat using a roller;

[0193] c) Place the glass fiber flat on a stainless steel mesh and dry overnight in a 37°C oven;

[0194] d) After taking out, dry and seal in the dark for storage for future use.

[0195] Test strip membrane drawing method:

[0196] In the present application, biotin is modified on the capture probes of the T line and the C line, and before being sprayed on the nitrocellulose membrane, streptavidin for membrane drawing is added to the detection capture probe, so that the biotin labeled on the capture probe spontaneously reacts with the streptavidin, so as to fix the detection capture probe on the nitrocellulose membrane, so as not to be washed away by the liquid.

[0197] The capture probe is modified with streptavidin by the following steps:

[0198] Mix 75 μL of capture probe modified with biotin (Jierui Biotechnology Co., Ltd.) at a concentration of 20 μM with 25 μL of streptavidin solution at a concentration of 1 mg / mL, and react at room temperature for 2 h to generate capture probe modified with biotin and streptavidin. Centrifuge the incubated sample at 6000 r / min at 4°C for 20 min, remove the precipitate, and store at 4°C for standby.

[0199] Draw the capture probe on the nitrocellulose membrane using a sputtering film drawing instrument to form a test line (T line); draw the quality control capture probe on the side close to the water absorption pad to form a quality control line (C line); dry the sputtered nitrocellulose membrane at 45°C overnight, and store in a dry room temperature environment away from light for standby.

[0200] Method for assembling the test strip:

[0201] Assemble the nitrocellulose membrane, sample pad, binding pad, and water absorption pad on the pad plate in sequence, with an overlap of 2 mm between adjacent two pads. After assembly, cut the test strip into 3.6 mm wide, and store in a dry room temperature environment away from light for standby.

[0202] Detection of Mycoplasma felis nucleic acid:

[0203] (1) Primer design

[0204] The present embodiment is a method for detecting pathogens by isothermal amplification, in which the product obtained by amplification with a primer DNA containing a T7 promoter and a specific pepper sequence is used as a template for synchronous T7 in vitro transcription. The full sequence of Mycoplasma felis is submitted to the database of the National Center for Biotechnology Information website for Blast search, and the homology is analyzed through the website program to select a sequence with low homology as a template sequence for primer design.

[0205] Template sequence:

[0206] Primer:

[0207] Primer F 5'-3'

[0208] Primer R 5'-3'

[0209] The nucleic acid sequence of the detection capture probe after modification with biotin is as follows:

[0210] T line capture probe:

[0211] C line capture probe:

[0212] Detection line and quality control line drawing method: after the probe is incubated with streptavidin, the film is drawn according to the above film drawing method, and the drawing amount is 1 μL / cm;

[0213] (2) Synthesis of template DNA:

[0214] The template DNA used in the application is a plasmid template composed of the full sequence of cat mycoplasma and pUC57 vector (purchased from Nanjing Kingsrui, item number C230KCMEG0-2, and the plasmid dilution step is strictly in accordance with the synthesis product use instruction of the company).

[0215] (3) Nucleic acid isothermal amplification:

[0216] Reaction reagent preparation:

[0217] The reaction reagent described here is applied in the isothermal amplification reaction process, and all solvents should be prepared and used immediately;

[0218] Reagent A:

[0219] Reagent B:

[0220] Isothermal amplification:

[0221] Add 14.7 μL of commercial kit (A buffer in Amply Future DNA isothermal rapid amplification kit (basic), item number 24041901C) to the enzyme freeze-dried reagent tube (purchased from Amply Future, stored in -20℃ refrigerator), and then add the following components in the tube in order:

[0222] After adding the components in the above table, cover the cap, mix well, and put 200 μL reaction tube into PCR instrument reaction, and the PCR instrument program (cap temperature 45℃) is set as follows:

[0223] (4) Amplification product detection

[0224] The test paper strip detection operation is as follows:

[0225] After the reaction tube is mixed well, 60 μL of the incubated sample is added to the sample well of the test paper card, and the test paper card is incubated in an incubator at 30°C for 5 min. After incubation, the test paper strip is irradiated under ultraviolet light, and the positive sample test paper strip has bright green T and C line fluorescent bands, while the negative sample test paper strip only has a bright green C line fluorescent band, as shown in FIG. 5A. Fluorescence reading is performed using a dry test paper strip reader, and the results are shown in FIG. 5B. The positive sample test paper strip has strong fluorescence signals at the T and C lines, while the negative sample test paper strip only has a strong fluorescence signal at the C line, and no fluorescence signal at the T line. This indicates that Pepper530 can be used as a reporter group on the addressable test paper strip for detecting targets in the amplified sample.

[0226] Example 2: Application of Pepper620 as a reporter group in lateral flow detection

[0227] The test paper strip is prepared according to the method in Example 1, and the sample is amplified according to Example 1. The sample is detected as follows:

[0228] After the reaction tube is mixed well, 60 μL of the incubated sample is added to the sample well of the test paper card, and the test paper card is incubated in an incubator at 30°C for 5 min. After incubation, the test paper strip is irradiated under ultraviolet light, and the positive sample test paper strip has bright green T and C line fluorescent bands, while the negative sample test paper strip only has a bright green C line fluorescent band, as shown in FIG. 5A. Fluorescence reading is performed using a dry test paper strip reader, and the results are shown in FIG. 5B. The positive sample test paper strip has strong fluorescence signals at the T and C lines, while the negative sample test paper strip only has a strong fluorescence signal at the C line, and no fluorescence signal at the T line. This indicates that Pepper530 can be used as a reporter group on the addressable test paper strip for detecting targets in the amplified sample.

[0229] Example 3: Application of silicon rhodamine nucleic acid aptamer as a reporter group and locked nucleic acid as a capture probe in lateral flow detection

[0230] Primer:

[0231] Forward primer 5'-3'

[0232] Reverse primer 5'-3'

[0233] I. Reaction system

[0234] Reaction system 1 (10 μL): 50 mM Tris-HCl, 35 mM KCl, 20 mM MgCl2, 4 mM NTPs, 1 mM dNTPs, 10% (v / v) glycerol, 15% (v / v) DMSO, 0.5 μM primer F (SEQ ID NO: 1), 0.5 μM primer R (SEQ ID NO: 5), SiR-NH2 (J. Am. Chem. Soc. 2019, 141, 7562-7571.) 1 μM.

[0235] Reaction system 2 (5 μL): 6.4 U AMV reverse transcriptase, 32 U T7 RNA polymerase, 0.08 U Rnase H, 2.1 μg BSA, 900 mM potassium sorbate.

[0236] II. Detection:

[0237] Amplification process: first mix the first stage reactants except the reaction enzyme, then add different amounts of new crown N gene RNA (SEQ ID NO: 33) (1 pM, 100 fM, 10 fM, no RNA (0)) and mix well, incubate at 65°C for 5 min, then continue to incubate at 43°C for 60 min.

[0238] The product analysis method used in the present application is lateral flow test strip detection. The test strip film and manufacturing method of the lateral flow test strip refer to Example 1.

[0239] T line capture probe: (the capture probe is synthesized by Shanghai Jeery Biological Engineering Co., Ltd.)

[0240] 5'-A+A+C+G+T+G+G+G+G+A+T+G+G+A+T+T+A+C+C+T+C+C+TTTTTTTTTT TTTTTTTT-biotin-3'; (here N+ indicates that the base is a locked nucleic acid (LNA), and N represents any base) (SEQ ID NO: 6)

[0241] C line capture probe:

[0242] The test strip detection operation is as follows (the operation is operated in the biological safety cabinet of the C level area): after the completion of amplification, 1 μL of SiR-NH2 dye DMSO solution is added to the reaction tube, so that the final concentration of the dye is 1 μM, and the system is fully mixed and then incubated at 50°C in a metal bath for 10 min. After incubation, cool to room temperature, take 60 μL of incubated sample and add it to the sample well of the test paper card, and incubate in a 30°C test paper card incubator for 5 min. After incubation, fluorescence reading is performed, and the results are shown in Table 1. The negative sample has almost no signal at the T line of the test strip, and the positive sample has a signal at the T line of the test strip. Moreover, as the target concentration increases, the fluorescence intensity at the T line continuously increases, and there is a strong fluorescence signal at the C line of the test strip, indicating that the silicon rhodamine aptamer can be used as a reporter group on the addressable test strip for detecting the target in the amplified sample. Moreover, within a certain range, the fluorescence intensity at the T line of the test strip has a positive correlation with the concentration of the target.

[0243] Table 1:

[0244] Example 4: Application of Clivia574 as a reporter group in lateral flow detection in the presence of a mediator probe

[0245] Forward primer 5'-3'

[0246] Reverse primer 5'-3'

[0247] The C line capture probe and the streptavidin modification method are as follows:

[0248] Mix 75 μL of biotin-modified C line capture probe (Jierui Biotechnology Co., Ltd.) with a concentration of 20 μM with 25 μL of streptavidin solution with a concentration of 1 mg / mL, and react at room temperature for 2 h to generate a capture probe modified with biotin and streptavidin. Centrifuge the incubated sample at 6000 r / min at 4°C for 20 min, remove the precipitate, and store at 4°C for later use.

[0249] The T line capture probe preparation method is as follows:

[0250] Prepare a 0.25 mg / mL streptavidin solution, centrifuge at 6000 r / min for 20 min, remove the precipitate, and store at 4°C for later use.

[0251] The nucleic acid sequence of the above detection capture probe after biotin modification is as follows:

[0252] Mediator probe:

[0253] C line capture probe:

[0254] The scribing was performed according to the method in Example 1, and the scribed large plate was dried in a 45°C oven overnight. The dried large plate was stored in a dry and dark environment for standby. The test strip was prepared according to the preparation method in Example 1.

[0255] Amplification product detection

[0256] The test strip detection operation is as follows:

[0257] The sample was amplified according to the method in Example 1. After the amplification was completed, 1 μL of NBSI574 dye DMSO solution was added to the reaction tube, so that the final concentration of the dye was 100 μM, and the mediator probe was added, so that the final concentration of the mediator probe was 200 nM. After mixing thoroughly, the system was incubated in a 50°C metal bath for 10 min. After incubation, it was cooled to room temperature, and 60 μL of the incubated sample was added to the sample well of the test strip card, which was incubated in a 30°C test strip card incubator for 5 min. After incubation, the fluorescence was read using a dry test strip reader. The results are shown in Figure 7. Under ultraviolet light, the positive sample test strip had bright orange T and C line fluorescence bands, while the negative sample test strip only had a bright orange C line fluorescence band without a T line band. This indicates that Clivia574 can be used as a reporter group, and the mediator probe can be used to detect the target in the amplified sample on the addressable test strip.

[0258] Example 5: Using bG aptamer as an example, the complex of ligand and dye is connected to construct a reporter group for addressable detection of target on a microfluidic chip

[0259] Primer:

[0260] Forward primer 5'-3'

[0261] Reverse primer 5'-3'

[0262] T line capture probe:

[0263] 5'-TFTFGFTFAFAFCFTFTFGFTFAFCFTFCFCFGFTFAFGFTTTTTTTTTTTTTTT-biotin-3'(SEQ ID NO:9)(Here NF represents that the ribose of the base is 2-F substituted, and N represents any base)

[0264] C line capture probe:

[0265] Microfluidic chip spotting:

[0266] Affinity modified microspheres (Suzhou Nanometer Technology Co., Ltd.) 100 μL (particle size 300 μm) were placed in a clean centrifuge tube, 500 μL of sterile ultrapure water was added, ultrasonic dispersion, 4°C, 14000 rap / min centrifugation, the supernatant was carefully removed, and the step was repeated once;

[0267] Microsphere coupling: The above washed microspheres were added to the PBS solution of the capture probe, so that the final concentration of the capture probe was 1 μM, then the system was placed on a rotary mixer, incubated at 4°C for 30 min, the supernatant was removed by centrifugation, 500 μL of resolvent was added to dissolve the precipitate, ultrasonic dispersion, 500 μL of sterile ultrapure water was added, ultrasonic dispersion, 4°C, 14000 rap / min centrifugation, the supernatant was carefully removed, and the step was repeated once, and stored at 4°C for standby;

[0268] The above labeled microspheres were drawn on the surface modified glass slide using a film drawing instrument, incubated in a constant temperature and humidity box overnight, washed with PBS the next day for three times, and dried at 30°C for 24 hours. The chip was assembled for standby;

[0269] The target sample and amplification method in Example 1 were used, except that the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 8) were used, and the bG and silicon rhodamine linked dye molecule (SNAP-Cell 647-SiR, NEB) was directly added before sample amplification. The final concentration of the dye molecule was 1 μM. After amplification, the sample was incubated on the microfluidic plate for 10 min, and the fluorescence intensity on the microfluidic plate was read using a microfluidic plate fluorescence reader. The results are shown in Table 2. The negative result has no fluorescence at the detection line, and the positive sample can clearly detect fluorescence at the detection line, indicating that the complex of bG aptamer as aptamer and ligand linked to dye as reporter group can be used for addressable detection of target on microfluidic chip.

[0270] Table 2:

[0271] Example 6: Using SRB-2 as aptamer and DN-PEG3-NH2 labeled color microspheres as labeled complex, the application of aptamer combined with ligand modified microspheres as reporter group in lateral chromatography detection was illustrated

[0272] DN-PEG3-NH2

[0273] Step 1:

[0274] 1.1 Microsphere washing: Take 100 μL of carboxylated microspheres (particle size 200 μm, blue, microsphere solid content 4 mg) (Suzhou Nanometer Technology Co., Ltd.) in a clean centrifuge tube, add 500 μL of sterile ultrapure water, ultrasonic dispersion, 4°C, 14000 rap / min centrifugation, carefully remove the supernatant, repeat the step 1 time;

[0275] 1.2 Microsphere activation: the microspheres in step 1.1 are dispersed in 4 mL of morpholine ethanesulfonic acid (MES) buffer solution with a concentration of 10 mmol, after ultrasonic dispersion, 0.2 mL of 5 mg / mL N-hydroxysulfosuccinimide (sμLfo-NHS) and 0.1 mL of 5 mg / mL 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) solution are added, and the carboxyl group is activated in a 37°C shaking bed for 2 h; 4°C, 8000 rpa / min-14000 rap / min centrifugation, carefully remove the supernatant, add 500 μL of sterile ultrapure water, ultrasonic dispersion, repeat the step 1 time;

[0276] 1.3 Microsphere coupling: add the amino ligand DN-PEG3-NH2 of the aptamer to the above activated microspheres, so that the final concentration of the amino ligand is 1 μM, then place the system in a rotary mixer, incubate at 4°C for 12 h, centrifuge and remove the supernatant, add 500 μL of resolvent to dissolve the precipitate, ultrasonic dispersion, add 50 μL of blocking solution containing 10% BSA and block for 1 h, centrifuge and wash, and store at 4°C for standby;

[0277] Step 2: Sample pad treatment: use the treatment solution for soaking treatment, then place it in a 37°C constant temperature drying box for drying for 2 h, and then perform the same spraying operation, wherein the treatment solution consists of Tris-HCl buffer with a working concentration of 0.05 M and pH=7.5-8.0, BSA with a mass concentration of 0.05%-0.5%, 1% sucrose and 0.05% Proclin300;

[0278] Step 3: Microsphere treatment of the binding pad: using a three-dimensional gold spraying point film instrument, the ligand-labeled microspheres prepared in step 1 are sprayed onto the glass fiber membrane at a rate of 10 μL / mL, and dried at 37°C for 12 h for standby;

[0279] Assemble the test strip according to the manner of Example 1, and card it for standby;

[0280] According to the sample amplification scheme in Example 1, amplification is performed, wherein the primers and probes are respectively:

[0281] Forward primer 5'-3'

[0282] Reverse primer 5'-3'

[0283] T line capture probe:

[0284] 5'-T+T+G+T+A+A+C+T+T+G+T+A+C+T+C+C+G+T+A+G+TTTTTTTTTTTTTT T-biotin-3' (SEQ ID NO: 11) (here N+ means the base 2-OMe modification, N represents any base)

[0285] C line capture probe:

[0286] Amplification product detection

[0287] The test strip detection operation is as follows:

[0288] According to the method of Example 1, sample amplification was carried out, except that the forward primer used was SEQ ID NO: 1, and the reverse primer was SEQ ID NO: 10. After amplification was completed, 60 μL of the sample after amplification was added to the sample well of the test strip card, and incubated in a test strip card incubator at 30°C for 5 min. After incubation was completed, fluorescence reading was performed using a dry test strip reader, and the results are shown in Figure 9 below. In the positive sample, clear T line and C line bands were observed, and in the negative sample, only the C line band was observed without the T line band, indicating that the SRB-2 aptamer and the DN-PEG3-NH2 labeled color microsphere reporter group can be used for addressable detection of the target on the test strip.

[0289] Example 7: Using SRB-2 as an aptamer and DN-PEG3-NH2 labeled HRP as a reporter group, the application of the aptamer conjugated ligand modified microsphere as a reporter group in lateral flow detection was demonstrated.

[0290] Step 1, weigh 4 mg of HRP dissolved in 0.2 mL of water (20 mg / mL), weigh NaIO4 37.9 mg dissolved in 1.895 mL of water (20 mg / mL), mix HRP and NaIO4 in a volume ratio of 1:1 (i.e. take 200 μL of each and mix slowly with stirring), and react at 4°C in the dark for 30 min. At this time, the solution is green.

[0291] Step 2, terminate oxidation

[0292] Add 4 μL of ethylene glycol to 40 μL of water, mix and then add to the oxidized HRP (slow stirring). React at 4°C in the dark for 30 min. At this time, the solution is brown.

[0293] Step 3, label ligand

[0294] The HRP solution terminated oxidation was directly added to the DN-PEG3-NH2, so that the final concentration of the DN-PEG3-NH2 was 400 μM, and the reaction was carried out at 4°C in the dark for 4 hours.

[0295] 5. Termination of labeling

[0296] 0.2 mg of NaBH4 was weighed and dissolved in 40 μL of water (freshly prepared and not stored), and all of the solution was added to the above reaction solution, and the reaction was carried out at 4°C in the dark for 1 hour, with shaking every 10 minutes.

[0297] 6. Purification and storage

[0298] 20 mM PBS (pH = 7.4) was dialyzed at 4°C in the dark overnight.

[0299] 7. The labeled protein in step 6 was treated with 2.5% w / v BSA and 1% w / v trehalose in PB (0.02 M, pH = 7.4) on a binding pad, and dried at 37°C overnight.

[0300] 8. The binding pad was cut to a width of about 0.7 cm, and the binding pad was attached to the nitrocellulose membrane, with a contact of about 2 mm, and the sample pad was attached to the binding pad, with a contact of about 2 mm, and the blotting paper was attached to the nitrocellulose membrane, with a contact of about 2 mm, and the strip was cut and ready for use.

[0301] 9. The amplification product in Example 6 was mixed with 3-amino-9-ethylcarbazole, so that the final concentration of 3-amino-9-ethylcarbazole was 1 mM, and 60 μL of the mixture was added to the sample pad of the test strip, and incubated at 30°C for 5 minutes, and the test results were observed, as shown in Figure 10. In the positive sample, clear red T and C line bands (the capture probe sequences of T and C lines are consistent with Example 6) were observed, and in the negative sample, only the red C line band was observed, without the T line band. This indicates that the complex of the aptamer ligand and the protein label can be used as a reporter group for the addressable detection of nucleic acid amplification samples.

[0302] Example 8: Addressable detection of amplification target on a gene chip using SRB-2 as an aptamer and DN-PEG3-NH2-labeled chemiluminescent group acridinone as a reporter group

[0303] Forward primer 5'-3'

[0304] Reverse primer 5'-3'

[0305] Capture probe:

[0306] Glass marking:

[0307] The amino glass slides were immersed in a 2.5% glutaraldehyde solution in PBS buffer (pH = 7.2) for 2 h at room temperature. After the reaction, the slides were washed three times with PBS buffer and three times with distilled water. The slides were left to dry at room temperature and were ready for use.

[0308] 1 μL of a 3 μM solution of the capture probe in water was added dropwise and the slides were incubated overnight at room temperature in a humidified environment. After incubation, the unbound oligonucleotide capture probe was removed by washing three times with TETBS (150 mM NaCl, 20 mM Tris, 5 mM EDTA, 0.05% (v / v) Tween 20, pH = 7.4) and TBS (150 mM NaCl, 20 mM Tris, pH = 7.4) solutions;

[0309] The slides were immersed in an aldehyde blocking solution (0.12 g of sodium borohydride dissolved in 30 mL of PBS, to which 10 mL of absolute ethanol were added, prepared extemporaneously and not for long-term storage) for 15 min. The slides must be completely immersed in the aldehyde blocking solution in order to block the aldehyde groups. The slides were immersed in 0.3 M glycine for 15 min.

[0310] The slides were washed again: twice for 2 min in 0.2% SDS and then twice for 2 min with deionized water;

[0311] NHS-activated acridinium ester (NSP-SA-NHS) (1.05 mM) was used with the DN-PEG3-NH2 ligand (1 mM) in 10 mL of anhydrous DMF, with the addition of anhydrous triethylamine (1.5 mM), under Ar protection, stirring at room temperature. After the reaction was complete, as determined by TLC, the solvent was removed under reduced pressure and the residue was purified by reverse-phase preparative chromatography, freeze-dried and stored at -20°C.

[0312] Sample amplification was performed according to the amplification method of Example 1, except that the forward primer used was SEQ ID NO: 1, the reverse primer was SEQ ID NO: 10, the amplified sample was mixed with the arylate-labeled ligand, the system was incubated at room temperature for 30 min, after incubation, TETBS (150 mM NaCl, 20 mM Tris, 5 mM EDTA, 0.05% (v / v) Tween 20, pH = 7.4) and TBS (150 mM NaCl, 20 mM Tris, pH = 7.4) solution were used for washing three times, 0.1 M sodium hydroxide solution and 0.1 M hydrogen peroxide solution were added, and imaging was performed using a gel imager in the dark, the results are shown in Figure 11, gray scale calculation was performed using ImageJ, the results are shown in Table 3, in the negative sample, the signal of the chip was weak, similar to the background fluorescence (taken at the edge of the slide), the positive sample detection position had obvious signal, the sample detection result was positive, and with the increase of the target concentration, the fluorescence at the chip detection point gradually increased, which indicated that the ligand-bound chemiluminescent molecule arylate as a reporter group could be used for addressable detection of amplified targets on the gene chip, and at the same time it also indicated that this method could be used for the detection of super-multiplex targets.

[0313] Table 3

[0314] Example 9: Application of Spinach-DFHBI as a reporter group in lateral flow detection

[0315] Forward primer 5'-3'

[0316] Reverse primer 5'-3'

[0317] T-line capture probe:

[0318] C-line capture probe:

[0319] 5'-A+T+A+A+A+G+T+G+T+A+A+T+G+T+T+A+C+T+AAAAAAAAAAAAAAA AA-biotin-3' (SEQ ID NO: 43); (here N+ represents that the base is 2-MOE modified, N represents any base)

[0320] The amplification mode and target concentration of Example 4 were used, except that the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 13) were used. Among them, DFHBI was added during amplification, and after amplification, the sample was added to the sample well of the microfluidic plate, incubated for 10 min, and the fluorescence intensity on the microfluidic plate was read using a microfluidic plate fluorescence reader. The results are shown in Table 4. The negative sample has almost no signal at the T line of the test strip, and the positive sample has a signal at the T line of the test strip. Moreover, with the increase of the target concentration, the fluorescence intensity at the T line is continuously enhanced, and there is a strong fluorescence signal at the C line of the test strip, indicating that Spinach-DFHBI can be used as a reporter group on the test strip with addressability to detect the target in the amplified sample, and within a certain range, the fluorescence intensity of the T line on the test strip has a positive correlation with the concentration of the target:

[0321] Table 4:

[0322] Example 10: Using Pepper599 as a reporter group and the SARS-CoV-2 nucleic acid sequence based on N gene as a target molecule, detecting the target molecule in the amplified product amplified by NASBA amplification technology on a lateral flow chromatogram

[0323] The sequence is as follows:

[0324] Primer:

[0325] Forward primer 5'-3':

[0326] Primer:

[0327] Reverse primer 5'-3':

[0328] I. Reaction system

[0329] Reaction system 1 (10 μL): 50 mM Tris, 35 mM KCl, 20 mM MgCl2, 4 mM NTPs, 1 mM dNTPs, 10% (v / v) glycerol, 15% (v / v) DMSO, 0.5 μM primer F (SEQ ID NO: 14), 0.5 μM primer R (SEQ ID NO: 15), HBC599 1 μM;

[0330] Reaction system 2 (5 μL): 6.4 U AMV reverse transcriptase, 32 U T7 RNA polymerase, 0.08 U Rnase H, 2.1 μg BSA, 900 mM potassium sorbate;

[0331] II. Detection

[0332] First, mix the first-stage reactants except the reaction enzyme, then add different amounts of new crown N gene RNA (1 pM, 10 fM, no RNA (0)), mix well, incubate at 65°C for 5 min, then continue to amplify at 43°C for 60 min;

[0333] The sequence of the new crown N gene is:

[0334] The test paper strip is made and the test paper strip is cut according to the test paper strip making method in Example 1;

[0335] T line capture probe:

[0336] C line capture probe:

[0337] The test paper strip detection operation is as follows:

[0338] After the amplification is completed, 1 μL of HBC599 dye DMSO solution is added to the reaction tube, so that the final concentration of the dye is 1 μM, and the system is mixed thoroughly and incubated at 50°C in a metal bath for 10 min. After incubation, cool to room temperature, take 60 μL of the incubated sample and add it to the sample well of the test paper card, and incubate it in a 30°C test paper card incubator for 5 min. After incubation, the test paper strip can see the positive sample test paper strip with bright red T line and C line fluorescent bands under ultraviolet irradiation, while the negative sample test paper strip only has bright red C line fluorescent band, and the T line band has no red fluorescent signal. The results are shown in Figure 12A. The fluorescence intensity of the test paper strip is read by using a dry test paper strip fluorescence reader. The negative sample can only read the fluorescence signal at the C line, and the positive sample can read the fluorescence signal at the T line and the C line respectively. With the increase of the target concentration, the fluorescence signal of the T line is significantly enhanced. The results are shown in Figure 12B, which shows that Pepper599 can be used as a reporter group to detect the target to be detected in the NASBA amplified sample on the test paper strip.

[0339] Example 11: Using SRB-2 as an aptamer and DN-PEG3-NH2 labeled methylene blue as a reporter group, the application of aptamer binding ligand modified electrochemical signal group as a reporter group on a gold electrode is illustrated

[0340] DN-PEG3-NH2 and NHS methylene blue are labeled and purified according to the method of Example 8, and are stored at -20°C in the dark for later use;

[0341] Primer:

[0342] Forward primer 5'-3'

[0343] Primer:

[0344] Reverse primer 5'-3':

[0345] T line capture probe:

[0346] The capture probe was dispersed in a Tris-TE buffer solution with a pH of 7 (20 mM Tris, 140 mmol NaCl and 5 mmol MgCl2 were added) and prepared to be 10 μM. 2 μL was added dropwise to a pretreated gold electrode, incubated at room temperature for 24 h, after the incubation was completed, washed three times with 10 mM Tris-HCl with a pH of 7.0, and stored at 4°C;

[0347] According to the amplification method of Example 1, except that the forward primer (SEQ ID NO: 17) and the reverse primer (SEQ ID NO: 15) were used. The amplified product was added dropwise to the electrode dried with nitrogen, incubated for 30 min, and the electrode was subjected to square wave voltammetry in the range of -0.35-1.5 V, and the results are shown in Figure 13. When the target was present, the electrode produced a clear electrochemical signal, and when the target was not present, no clear electrochemical signal was detected, as shown in Figure 13, indicating that the aptamer binding ligand modified electrochemical signal group can be used as a reporter group for the addressable detection of the amplified target.

[0348] Example 12: Detection of the target content of miRNA-21 in the rolling circle amplification detection system by using fluorescent RNA Peppe530 as a reporter group

[0349] As shown in Figure 14A, the probe structure inserts detection sequences, random sequences, T7 promoter recognition sequences, etc. The designed circular template mainly includes a target recognition region, a promoter region, a fluorescent RNA region, etc. The amplification schematic diagram is shown in Figure 14B.

[0350] The sequences of the probes and the like are as follows:

[0351] Detection probe sequence: 5'-3'

[0352] T7 promoter sequence: 5'-3'

[0353] Capture probe sequence: 5'-3'

[0354] According to the method of Example 8, a gene chip was prepared and stored at 2-8°C for standby;

[0355] (2) Synthesis of detection probe

[0356] The detection probe in the present application is a long single-stranded DNA, and a phosphorylation modification needs to be added to the 5' end (purchased from Nanjing Kingsrui, and the dilution step of the primer is performed according to the usage instruction of the synthesized product of the company).

[0357] (3) Nucleic acid isothermal amplification:

[0358] Preparation of reaction buffer (Reaction Buffer) reaction solution:

[0359] The reaction solution described here should be prepared and used immediately, with pH = 7.4 and temperature of 25°C.

[0360] Isothermal amplification: the following components are sequentially added in the tube:

[0361] After adding, place it in a temperature control instrument (such as a PCR instrument) and perform the reaction according to the following program:

[0362] Drop the amplification product on the chip capture area, repeat 3 points for each sample concentration, incubate at room temperature for 2h, after incubation, wash with TETBS (150mM NaCl, 20mM Tris, 5mM EDTA, 0.05% (v / v) Tween 20, pH = 7.4) and TBS (150mM NaCl, 20mM Tris, pH = 7.4) solution three times, use a microscope to image each sample pad, the results are shown in FIG. 14C, use ImageJ to calculate the gray scale, the results are shown in FIG. 14D, in the negative sample, the signal of the chip is weak, the positive sample detection position has obvious signal, the sample detection result is positive, and with the increase of the target concentration, the fluorescence at the chip detection point gradually increases, and increases with the increase of the target concentration, which shows that the fluorescent RNA as a reporter group can be used for addressable detection of RCA amplification target on the gene chip, and at the same time it also shows that this method can be used for the detection of different targets by fixing the capture probe at different positions, thereby for the detection of super-multiplex targets.

[0363] Example 13: Use of fluorescent DNA as a label for multiplex detection of HPV on lateral flow test strips

[0364] According to the above principle, we first designed a detection method to detect five common HPV types, HPV 6, 11, 16, 18 and 33, and human beta globin (HBB) gene as an internal positive control (IPC) to avoid potential false negatives. Universal primers GP5+ (SEQ ID NO: 22) / GP6+ (SEQ ID NO: 23) were used for amplification, and the amplification system is shown in the following table:

[0365] Template sequence:

[0366] HPV6:

[0367] HPV18:

[0368] HPV-16:

[0369] HPV-33:

[0370] HPV-11:

[0371] PCR system:

[0372] PCR reaction condition:

[0373] GP5+ primer:

[0374] GP6+ primer:

[0375] IC primer:

[0376] IC forward primer sequence:

[0377] IC reverse primer sequence:

[0378] Capture probe:

[0379] HPV6:

[0380] HPV11:

[0381] HPV16:

[0382] HPV18:

[0383] HPV33:

[0384] C-line capture probe:

[0385] Incubate the probe with streptavidin, draw on the T1, T2, T3, T4, T5 and C-line positions of the NC membrane respectively, and assemble the test strip in the manner of Example 1;

[0386] HPV test type is used as a positive sample, which is a standard synthetic type containing a plasmid containing a GP5+ / GP6+ flanking region, each containing an HPV type (HPV6, HPV11, HPV16, HPV18, HPV33), the positive sample contains the above synthetic sample, and the negative sample uses only an equal volume of normal saline, wherein the negative sample and the positive sample are added with the HBB gene, and the amplification is carried out according to the components and amplification mode in the above table, and the product after amplification is incubated with the dye to make the final concentration of the dye 1 μM, 100 μL of the incubated product is added to the test strip, and lateral chromatography is carried out at 30°C, and the test strip after incubation can be observed under the irradiation of an ultraviolet lamp or the like. The test strip of the positive sample has 5 bright green T lines and 1 bright green C line fluorescent band, while the test strip of the negative sample has only one bright green C line fluorescent band, as shown in FIG. 15A. The fluorescence reading is carried out using a dry test strip reading instrument, and the results are shown in FIG. 15B. The 5 T lines and C lines on the test strip of the positive sample have strong fluorescence signals, while the test strip of the negative sample has only a strong fluorescence signal on the C line, and no fluorescence signal on the T line, indicating that the fluorescent DNA can be used as a reporter group on the test strip with addressability for simultaneous detection of multiple targets in the amplified sample.

[0387] Example 14: Okra as a reporter group, application of super-multiplex target detection in lateral chromatography detection

[0388] The film is cut and the test strip is prepared in the manner of Example 1.

[0389] Feline mycoplasma nucleic acid detection:

[0390] (1) Primer design

[0391] This example is a method for detecting super-multiplex pathogens by isothermal amplification, in which the product obtained by amplification with primer DNA containing a T7 promoter and a specific Okra sequence is used as a template for synchronous T7 in vitro transcription. The specific target sites of influenza A virus, rhinovirus B, respiratory syncytial virus type B, influenza B virus, respiratory syncytial virus type A, adenovirus, bocavirus, human metapneumovirus, parainfluenza virus I, parainfluenza virus II, parainfluenza virus III, and parainfluenza virus IV are selected as template sequences for primer design.

[0392] Detection line and quality control line drawing method: After incubation of the probe with streptavidin, the film is cut according to the above film cutting method, and the amount of drawing is 1 μL / cm;

[0393] (2) Template DNA synthesis:

[0394] The template DNA used in the present application is a plasmid template composed of a target sequence and a pUC57 vector (purchased from Nanjing Kingsway, Influenza A virus, item number C5549621G0-2, Rhinovirus B, item number C048J166G0-2, Respiratory Syncytial Virus B, item number C048J166G0-4, Influenza B virus, item number C048J166G0-6, Respiratory Syncytial Virus A, item number C9112BZNG0-2, Adenovirus, item number C092WFMPG0-2, Bocavirus, item number C485T957G0-2, Human Metapneumovirus, item number C6866NGDG0-2, Parainfluenza Virus I, item number C707TCNPG0-1, Parainfluenza Virus II, item number C707TCNPG0-4, Parainfluenza Virus III, item number C707TCNPG0-6, Parainfluenza Virus IV, item number C707TCNPG0-8, and the plasmid dilution step was strictly in accordance with the product use instructions of the company).

[0395] (3) Nucleic acid isothermal amplification:

[0396] Preparation of reaction reagents:

[0397] The reaction reagents described herein are applied in the present isothermal amplification reaction process, and all solvents should be prepared and used immediately;

[0398] Reagent A:

[0399] Reagent B:

[0400] Isothermal amplification:

[0401] In the enzyme freeze-dried reagent tube (purchased from Ampure Future and stored in a -20°C refrigerator), 14.7 μL of A buffer was added, and then the following components were added in order in the tube:

[0402] After adding the components in the above table, cover the lid and mix, place 200 μL of the reaction tube into the PCR instrument reaction, and set the PCR instrument program (lid temperature 45°C) as follows:

[0403] (4) Amplification product detection

[0404] The test paper strip detection operation is as follows:

[0405] In the reaction tube after amplification is completed, 1 μL ACE dye DMSO solution is added to make the final concentration of the dye 3.5 μM, and the system is mixed thoroughly. 100 μL of the incubated sample is added to the sample well of the test paper card, and placed in a 37°C test paper card incubator for incubation for 15 min. After incubation, the test paper strip of the positive sample can be seen under the irradiation of ultraviolet light, etc. There are bright green T line and C line fluorescent bands, while the negative sample has only bright green C line fluorescent band without T line band, as shown in FIGS. 16A and 16B. The dry test paper strip reader is used for fluorescence reading, and the results are shown in FIG. 16D. The test paper strip of the positive sample has strong fluorescence signals of T line and C line, while the test paper strip of the negative sample has strong fluorescence signal of C line and no fluorescence signal of T line, as shown in FIG. 16C. It is shown that Okra can be used as a reporter group on the addressable test paper strip for detecting targets in the super-multiplex amplification sample.

Claims

1. A method for addressable detection of a nucleic acid target, characterized in that, The method comprises: a) preparing aptamer-target units by amplification, wherein the aptamer-target units comprise one or more aptamers and one or more nucleic acid targets bound to the one or more aptamers; b) preparing a solid support and one or more capture elements immobilized on the surface and / or inside of the solid support, which can specifically capture the aptamer-target units; c) a label that can specifically recognize and / or bind to the aptamer-target units, the aptamer-target units and the capture elements form a complex, wherein the label has a detectable signal after binding to the aptamer-target units; and d) detecting the signal.

2. The method of claim 1, wherein, The amplification is a variable temperature amplification or an isothermal amplification.

3. The method of claim 1, wherein, The aptamer and the nucleic acid target are on the same nucleic acid strand; preferably, the aptamer and the nucleic acid target are directly connected, the aptamer and the nucleic acid target are operably linked by one or more nucleotide sequences, or the aptamer and the nucleic acid target comprise one or more shared nucleotide sequences; Alternatively, the aptamer and the nucleic acid target are not on the same nucleic acid strand; preferably, the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are independently amplified in the same system or mixed after amplification in different systems; more preferably, the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are hybridized and combined by affinity, covalent bond or base complementary pairing.

4. The method of claim 1, wherein, The aptamer is selected from a DNA aptamer or an RNA aptamer; Preferably, when the aptamer is a DNA aptamer, the aptamer is a non-G-quadruplex; More preferably, the length of the aptamer is 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further more preferably 10-200 nt, most preferably 10-100 nt; Further more preferably, the aptamer is selected from one or a combination of Pepper or its mutants, Clivias or its mutants, Mango or its mutants, Spinach or its mutants, Broccoli or its mutants, BiRhoBAST or its mutants, biSiRA or its mutants, Riboglow RNA tags or its mutants, Chili or its mutants, Okra or its mutants, o-Coral or its mutants, DIR2s-apt or its mutants, 13-2min or its mutants, and MGA or its mutants.

5. The method of claim 1, wherein, The label comprises a ligand that specifically recognizes and / or binds to the aptamer-target unit; Preferably, the label further comprises a reporter group connected to the ligand, and the reporter group is selected from one or a combination of dyes, nanoparticles, microspheres, redox molecules, luminescent molecules, radioactive labels, electrochemical functional groups, enzymes and enzymes plus detectable enzyme substrates.

6. The method of claim 1, wherein, The label has a detectable signal before binding to the aptamer-target unit and the label has a detectable signal after binding to the aptamer-target unit with different intensities; More preferably, when the detectable signal is a fluorescent signal, the fluorescent signal intensity of the label after binding to the aptamer-target unit is enhanced by more than 5 times, preferably the fluorescent signal intensity is enhanced by more than 10 times or the excitation wavelength shift is more than 10 nm or the emission wavelength shift is more than 10 nm, more preferably the fluorescent signal intensity is enhanced by more than 20 times or the excitation wavelength shift is more than 20 nm or the emission wavelength shift is more than 20 nm, further preferably the fluorescent signal intensity is enhanced by more than 50 times or the excitation wavelength shift is more than 50 nm or the emission wavelength shift is more than 50 nm, compared to the fluorescent signal intensity of the label before binding to the aptamer-target unit; Further more preferably, the label forms a covalent or non-covalent complex with the aptamer-target unit; when the label forms a non-covalent complex with the aptamer-target unit, the dissociation constant (Kd) between the label and the aptamer-target unit is selected from the range of about 0.001 nM to about 100 μM, preferably from the range of about 0.01 nM to about 1 μM, more preferably from the range of about 0.01 nM to about 10 nM; Most preferably, the complex formed by the label and the aptamer-target unit is selected from one or a combination of Pepper485, Pepper497, Pepper508, Pepper514, Pepper525, Pepper530, Pepper599, Pepper620, Clivia580, Clivia577, Clivia581, Clivia582, Clivia590, Clivia600, Clivia624, Clivia565, Clivia570, Clivia571, Clivia574, Clivia578, Clivia595, Clivia618, Chili-DMHBI-Imi and Chili-DMHBO.

7. The method of claim 1, wherein, The aptamer-target unit binds to the label to form the complex before binding to the capture element; Alternatively, the aptamer-target unit binds to the capture element to form the complex before binding to the label; Alternatively, the process of the aptamer-target unit binding to the label and the capture element to form the complex is carried out simultaneously.

8. The method of claim 1, wherein, The capture element is selected from one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, morpholino backbone nucleic acid, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cell receptor, ligand, lipid, biotin, avidin, streptavidin, exavidin, neutravidin, Traptavidin, metal and histidine; preferably one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, antigen, antibody, aptamer or biotin; more preferably one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence or biotin; More preferably, when the capture element is a nucleic acid sequence, the nucleic acid sequence comprises at least one modification, and the modification is independently at one or more selected from the group consisting of ribose position, deoxyribose position, phosphate position, and base position; Further more preferably, the modification is at the phosphate position of the aptamer, and the modification is a modification to resist nuclease activity selected from one or a combination of phosphorothioate linkage, alkyl phosphorotriester linkage, aryl phosphorotriester linkage, alkyl phosphorothioate linkage, aryl phosphorothioate linkage, hydrogen phosphorothioate linkage, and alkyl aminophosphorothioate linkage; or the modification is at the ribose position of the aptamer, and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analogue of a mirror body; or the modification is at the base position of the aptamer, and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

9. The method of claim 1, wherein, The capture element specifically captures the aptamer-target unit is by affinity capture, covalent reaction, hybridization capture by base complementarity, capture of antibody and antigen / hapten, capture of chemical tag and substrate, capture of enzyme and substrate, capture of nanoenzyme and substrate, or mediator probe; More preferably, the mediator probe comprises a probe region and a capture binding region; wherein the probe region has affinity to the aptamer-target unit, the capture binding region has recognition binding force to the capture element, the probe region and the capture binding region are independently selected from one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cell receptor, ligand, lipid, biotin, avidin, streptavidin, neutravidin, Traptavidin, metal, and histidine, preferably selected from one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, antigen, antibody, aptamer, and biotin, more preferably selected from one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, and biotin; Further more preferably, the mediator probe further comprises an intermediate connecting region; wherein the intermediate connecting region is selected from one or a combination of nucleoside, peptide chain, peptide nucleic acid chain, monosaccharide, polysaccharide, PEG chain, high molecular polymer, short chain chemical linker, and chain segment.

10. A kit for the addressable detection of a nucleic acid target, characterized in that, The kit comprises a primer set for amplification of the aptamer-target unit, a label, a solid support, a capture element, and optionally an instruction of detection method; The primer set comprises primers for amplification of one or more nucleic acid targets and the 5' end of the primers further comprises an aptamer coding sequence, or the primer set comprises primers for amplification of one or more nucleic acid targets and primers for amplification of one or more aptamers; The aptamer-target unit is formed by variable temperature amplification or isothermal amplification; the label has a detectable signal after binding to the aptamer-target unit; the capture element is immobilized on the surface or inside of the solid support and can specifically capture the aptamer-target unit.

11. The kit of claim 10, wherein The aptamer and the nucleic acid target are located on the same nucleic acid strand after amplification; preferably, the aptamer and the nucleic acid target are directly connected, the aptamer and the nucleic acid target are operably linked by one or more nucleotide sequences, or the aptamer and the nucleic acid target comprise one or more shared nucleotide sequences; Alternatively, the aptamer and the nucleic acid target are not located on the same nucleic acid strand after amplification; preferably, the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are independently amplified in the same system or are mixed after amplification in different systems; more preferably, the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are combined by affinity or covalent bond, or the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are combined by base complementary pairing and hybridization.

12. The kit of claim 10, wherein The aptamer is selected from a DNA aptamer or an RNA aptamer; Preferably, when the aptamer is a DNA aptamer, the aptamer is a non-G-quadruplex; More preferably, the length of the aptamer is 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further more preferably 10-200 nt, most preferably 10-100 nt; Further more preferably, the aptamer is selected from one or a combination of Pepper or its mutant, Clivias or its mutant, Mango or its mutant, Spinach or its mutant, Broccoli or its mutant, BiRhoBAST or its mutant, biSiRA or its mutant, Riboglow RNA tags or its mutant, Chili or its mutant, o-Coral or its mutant, DIR2s-apt or its mutant, 13-2min or its mutant, and MGA or its mutant, Okra or its mutant.

13. The kit of claim 10, wherein The label comprises a ligand that specifically recognizes and / or binds to the aptamer-target unit; Preferably, the label further comprises a reporter group connected to the ligand, and the reporter group is selected from one or a combination of dyes, nanoparticles, microspheres, redox molecules, luminescent molecules, radioactive labels, electrochemical functional groups, enzymes, and enzymes plus detectable enzyme substrates.

14. The kit of claim 10, wherein the detectable signal of the label before binding with the aptamer-target unit is different from the detectable signal of the label after binding with the aptamer-target unit; more preferably, when the detectable signal is a fluorescent signal, the fluorescent signal of the label after binding with the aptamer-target unit is enhanced by more than 5 times, preferably the fluorescent signal is enhanced by more than 10 times or the excitation wavelength shift is more than 10 nm or the emission wavelength shift is more than 10 nm, more preferably the fluorescent signal is enhanced by more than 20 times or the excitation wavelength shift is more than 20 nm or the emission wavelength shift is more than 20 nm, further preferably the fluorescent signal is enhanced by more than 50 times or the excitation wavelength shift is more than 50 nm or the emission wavelength shift is more than 50 nm, compared to the fluorescent signal of the label before binding with the aptamer-target unit; more preferably, the label forms a covalent or non-covalent complex with the aptamer-target unit; when the label forms a non-covalent complex with the aptamer-target unit, the dissociation constant (Kd) between the label and the aptamer-target unit is selected from the range of about 0.001 nM to about 100 μM, preferably from the range of about 0.01 nM to about 1 μM, more preferably from the range of about 0.01 nM to about 10 nM; most preferably, the complex formed by the label and the aptamer-target unit is selected from one or a combination of Pepper485, Pepper497, Pepper508, Pepper514, Pepper525, Pepper530, Pepper599, Pepper620, Clivia580, Clivia577, Clivia581, Clivia582, Clivia590, Clivia600, Clivia624, Clivia565, Clivia570, Clivia571, Clivia574, Clivia578, Clivia595, Clivia618, Chili-DMHBI-Imi and Chili-DMHBO.

15. The kit of claim 10, wherein the capture element is selected from one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, morpholino backbone nucleic acid, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cell receptor, ligand, lipid, biotin, avidin, streptavidin, extravidin, neutravidin, Traptavidin, metal and histidine; preferably one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, antigen, antibody, aptamer or biotin; more preferably one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence or biotin; more preferably, when the capture element is a nucleic acid sequence, the nucleic acid sequence comprises at least one modification, and the modification is independently at one or more selected from the group consisting of ribose position, deoxyribose position, phosphate position and base position; Further more preferably, the modification occurs at a phosphate position of the aptamer, and the modification is a modification conferring resistance to nuclease activity, the modification being selected from one or a combination of phosphorothioate linkage, alkyl phosphorotriester linkage, aryl phosphorotriester linkage, alkyl phosphorothioate linkage, aryl phosphorothioate linkage, hydrogen phosphorothioate linkage, and alkyl phosphoramidate linkage; or the modification occurs at a ribose position of the aptamer, and the modification is selected from one or a combination of 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro (2'-F), 2'-methoxy (2'-OMe), 2'-ethoxy (2'-OEt), 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O-butyl modification, 1-(4'-thio-PD-ribofuranose) modification, 2-O-4-C modification, L-DNA, and a nucleic acid analogue of a mirror body; or the modification occurs at a base position of the aptamer, and the modification is selected from one or a combination of 5'-position pyrimidine modification, 8'-position purine modification, cytosine exocyclic modification, substitution of 5'-bromouracil, substitution of 5'-bromodeoxyuridine, substitution of 5-bromodeoxycytidine, backbone modification, methylation, 2'-methoxy ethylene (2'-MOE), 3' cap, and 5' cap.

16. The kit of claim 10, wherein The kit further comprises a mediator probe, the mediator probe comprising a probe region and a capture binding region; wherein the probe region has affinity for the aptamer-target unit, the capture binding region has a recognition binding force for the capture element, the probe region and the capture binding region are independently selected from one or a combination of natural nucleic acid sequence, unnatural nucleic acid sequence, peptide nucleic acid, antigen, antibody, hapten, enzyme, nanoenzyme, aptamer, monosaccharide, polysaccharide, affibody, antibody mimetic, cell receptor, ligand, lipid, biotin, avidin, streptavidin, exavidin, neutravidin, Traptavidin, metal, and histidine, preferably one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, antigen, antibody, aptamer, and biotin; more preferably one or a combination of peptide nucleic acid, natural nucleic acid sequence, unnatural nucleic acid sequence, and biotin; More preferably, the mediator probe further comprises an intermediate linking region; wherein the intermediate linking region is selected from one or a combination of nucleoside, peptide chain, peptide nucleic acid chain, monosaccharide, polysaccharide, PEG chain, high molecular polymer, short chain chemical linking group, and chain segment.

17. An aptamer-target unit for addressable detection of a nucleic acid target, characterized in that, The aptamer-target unit comprises one or more aptamers and one or more nucleic acid targets bound to the one or more aptamers.

18. The aptamer-target unit of claim 17, wherein, The aptamer and the nucleic acid target are located on the same nucleic acid strand; preferably, the aptamer and the nucleic acid target are directly connected, the aptamer and the nucleic acid target are operably linked by one or more nucleotide sequences, or the aptamer and the nucleic acid target comprise one or more shared nucleotide sequences; Alternatively, the aptamer and the nucleic acid target are not on the same nucleic acid strand; preferably, the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are bound by affinity or covalent bond, or the nucleic acid strand where the aptamer is located and the nucleic acid strand where the nucleic acid target is located are bound by base complementary pairing hybridization.

19. The aptamer-target unit of claim 17, wherein, The aptamer is selected from a DNA aptamer or an RNA aptamer; Preferably, when the aptamer is a DNA aptamer, the aptamer is a non-G-quadruplex; More preferably, the length of the aptamer is 10-500 nt, preferably 10-300 nt, more preferably 10-250 nt, further more preferably 10-200 nt, most preferably 10-100 nt; Further more preferably, the aptamer is selected from one or a combination of Pepper or its mutants, Clivias or its mutants, Mango or its mutants, Spinach or its mutants, Broccoli or its mutants, BiRhoBAST or its mutants, biSiRA or its mutants, Riboglow RNA tags or its mutants, Chili or its mutants, o-Coral or its mutants, DIR2s-apt or its mutants, 13-2min or its mutants, and MGA or its mutants, Okra or its mutants.

20. Use of the aptamer-target unit of claims 17-19 or the kit of claims 10-16 in the detection of addressability of a nucleic acid target.

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