A DNA probe system and method for aptamer-driven cyclic detection of kanamycin based on G-tetramer response

Abstract

The present invention provides a DNA probe system and method for detecting kanamycin based on an aptamer-driven cyclic detection method based on G-tetramer response, belonging to the field of bioanalysis and detection technology. The system utilizes the specific recognition of aptamers and the cyclic reuse of probes initiated by nuclease III, combined with a toehold-mediated specific chain displacement reaction, to achieve target signal amplification detection. The split-type G-tetramer is used as a chimeric receptor that specifically illuminates the ThT signal molecule. When there is no target activation cycle to produce RP for downstream chain displacement reaction, signal transduction cannot be achieved, thereby significantly reducing background signals and improving signal-to-time ratios. At the same time, a split-type G-Quadruplex is used to construct a ThT label-free signal output model to achieve specific and highly sensitive detection of the target kana.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and particularly relates to a DNA probe system and method for detecting kanamycin in a cyclic manner driven by an aptamer based on G-tetramer response. Background Art

[0002] The discovery and application of antibiotics marked a major breakthrough in human medicine, significantly reducing mortality from infectious diseases. However, with widespread use, the problem of antibiotic misuse has gradually evolved into a global public health crisis. The most serious consequence of antibiotic misuse is the emergence of drug-resistant bacteria, also known as "superbugs." According to a study published in The Lancet, in 2019, 1.27 million people worldwide died directly from drug-resistant infections, and 4.95 million deaths were related to them. It is predicted that by 2050, drug resistance will cause 10 million deaths annually worldwide, with direct economic losses reaching $100 trillion. Antibiotic misuse can also lead to the emergence of drug-resistant genes, causing cross- and multidrug resistance in organisms, posing a threat to human life.

[0003] Kanamycin (Kana), a protein biosynthesis inhibitor, is produced by Streptomyces kanamycinii. Since its isolation from Streptomyces kana in 1957, it has become a major representative of the amino acid glycoside class of proteins. It is widely used in veterinary medicine to inhibit the growth of both Gram-positive and Gram-negative bacteria and is an indispensable antimicrobial agent in the treatment of poultry and livestock diseases. In swine farming, this drug has a cure rate of over 85% for endemic pneumonia, but excessive use can also pose a significant risk of residual disease. Improper use of Kana can lead to excessive antibiotic residues in animal-derived foods and can also cause other problems in humans besides allergies, such as gastrointestinal dysbiosis and nephrotoxicity. Furthermore, long-term use of kanamycin may result in high and persistent tissue residues due to its tissue affinity.

[0004] Currently, Kana detection technologies primarily include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). However, these mainstream methods all have significant shortcomings: ELISA antibody reagents suffer from poor stability during room-temperature transportation and exhibit a high coefficient of variation; HPLC equipment is expensive to purchase and requires specialized mass spectrometry personnel, making Kana detection expensive; and CE technology has complex pretreatment procedures, lengthy testing times, and limited timeliness. Therefore, establishing a rapid, convenient, and low-cost method for sensitively detecting Kana residues in food is of crucial practical significance.

[0005] The emergence of DNA nanotechnology represents a revolutionary breakthrough in the field of artificially controlled molecular self-assembly, with profound implications for nanoscience and nanotechnology. Through sequence design, linear DNA molecules can self-assemble into a variety of two- or three-dimensional nanostructures, exhibiting a high degree of order, controllability, addressability, and stability. With the development of DNA nanotechnology, dynamic DNA nanotechnology has developed a new reaction based on DNA hybridization: the toehold-mediated DNA strand displacement reaction, which can achieve DNA structural reconstruction and dynamic equilibrium in molecular devices. A DNA strand displacement reaction occurs when a hybridized double-stranded DNA strand can be displaced by another nucleic acid through partial or complete base pairing. The strand displacement reaction can only proceed when the recognition sequence is completely correct. Therefore, this toehold-mediated strand displacement reaction ensures high specificity, providing new opportunities for precise DNA assembly.

[0006] DNA G-quadruplexes are composed of guanine-rich nucleic acid sequences. Stabilized by specific cations such as K+ and Na+, they can form multiple G-quadruplexes connected by Hoogsteen hydrogen bonds and further stacked into a unique secondary structure. These structures can form a variety of topological structures, including parallel, antiparallel, and hybrid structures, through intra- and inter-strand stacking. Thioflavin T (ThT) is a water-soluble fluorescent dye that, in solution, emits only weak or no fluorescence due to exciton quenching caused by intramolecular motion. However, upon interaction with DNA, it preferentially binds to G-quadruplex structures. In the aggregated state, due to restricted intramolecular motion, nonradiative transitions are suppressed, resulting in significant fluorescence enhancement. Leveraging its "light-up" properties, G-quadruplex / ThT can be used as a molecular signal to construct label-free fluorescent biosensing strategies for target detection.

[0007] Aptamers are oligonucleotide sequences with high specificity and affinity for their target substances, obtained by screening from random oligonucleotide libraries using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. Aptamers can form stable complexes with their target substances. The emergence of aptamers has opened up a new dimension in biosensing technology, with their target recognition mechanism transcending the limitations of traditional antibodies. Compared to other molecular recognition components commonly used in biosensing technologies, aptamers offer greater biostability, ease of preparation, excellent specificity, high affinity, and the flexibility to modify a variety of groups. Summary of the Invention

[0008] In order to solve the above technical problems, this solution provides an aptamer-driven cyclic detection DNA probe system and method for kanamycin based on G-tetramer response. It aims to utilize the specific recognition of aptamer and target and the DNA toehold-mediated chain displacement reaction, combined with the hydrolysis of nuclease III and the specific binding of ThT to split-G-Quadruplex, to construct an aptamer-driven cyclic DNA biosensor to achieve label-free, low background and highly sensitive detection of kanamycin.

[0009] To achieve the above objectives, this solution first provides an aptamer-driven cyclic detection DNA probe system for kanamycin based on G-tetramer response. The system includes probe D1, probe D2, hairpin probe H1, probe BG, and nuclease III. Probes D1 and probe D2 self-assemble into a double-stranded probe through base complementary pairing. Probe BG is self-assembled by probes B1, probe B2, probe G4-a, and probe G4-b through base complementary pairing. In the presence of kanamycin, kanamycin is specifically recognized by probe D1, and detection of kanamycin is achieved through multiple signal amplification.

[0010] The sequence of the probe D1 is shown in SEQ ID NO. 1;

[0011] The sequence of the probe D2 is shown in SEQ ID NO. 2;

[0012] The sequence of the hairpin probe H1 is shown in SEQ ID NO. 3;

[0013] The sequence of the probe B1 is shown in SEQ ID NO. 4;

[0014] The sequence of the probe B2 is shown in SEQ ID NO. 5;

[0015] The sequence of the probe G4-a is shown in SEQ ID NO. 6;

[0016] The sequence of the probe G4-b is shown in SEQ ID NO. 7.

[0017] Based on a general inventive concept, this solution also provides a method for detecting kanamycin using a DNA probe system for non-diagnostic purposes, comprising the following steps:

[0018] S1. DNA probe pretreatment: Prepare lyophilized powders of synthesized probes D1, D2, H1, B1, B2, G4-a, and G4-b into 100 µM stock solutions with DEPC water and store at 4°C until use.

[0019] Construction of double-stranded probes S2 and D1 / D2: Probes D1 and D2 were added to a PBS buffer at pH 7.4 at a molar ratio of 1:1. The probes were annealed at 95°C for 5 min and then slowly cooled to allow them to self-assemble into double-stranded probes D1 / D2 through complementary base pairing. The probes were then stored in a refrigerator at 4°C until use.

[0020] S3. Annealing of hairpin probe H1: Anneal the hairpin probe H1 at 95°C for 5 min and then slowly cool it to allow it to self-assemble into a hairpin structure through base complementary pairing. Store it in a refrigerator at 4°C until use.

[0021] S4. Construction of probe BG: Probe B1, probe B2, probe G4-a, and probe G4-b were added to PBS buffer at pH 7.4 at a molar ratio of 1:1:1:1. The mixture was annealed at 95°C for 5 min and then slowly cooled to allow the probes to self-assemble into probe BG through complementary base pairing. The probes were then stored in a refrigerator at 4°C until use.

[0022] S5. Upstream target activation: The sample to be tested is incubated with 1 µM D1 / D2 double-stranded probe and hairpin probe H1, and exonuclease III is added to release probe D1 for recycling;

[0023] S6, downstream competitive reaction: 1 μM BG probe constructed by annealing in S4 was added to the system prepared in S5 and incubated together, and K + , forming a DNA tetramer structure for signal activation;

[0024] Signal activation and fluorescence detection of S7, G tetramer-thioflavin T: 40 μM thioflavin T was added to the system prepared by S6, and the reaction was carried out at 37°C in PBS buffer for 30 min. The fluorescence spectrum was collected using an RF-6000 fluorescence spectrophotometer.

[0025] Preferably, the incubation time in step S5 is 30 min and the incubation temperature is 37°C.

[0026] Preferably, the incubation time in step S6 is 30 min and the incubation temperature is 37°C.

[0027] Preferably, the pH of the PBS buffer in step S7 is 7.4 and includes 50 mM KCl.

[0028] Preferably, in step S7, the fluorescence spectrophotometer is set to an excitation wavelength Ex=450 nm, an emission wavelength Em=465-600 nm, and the excitation and emission slit widths are set to 5 nm and 5 nm, respectively.

[0029] The mechanism of miRNA detection provided by this protocol is as follows:

[0030] This scheme designs Aptamer-functionalized double-stranded probes D1 / D2. When the target Kana is present, Kana specifically recognizes the Aptamer sequence D2, releasing the complementary sequence D1. D1 can specifically bind to the hairpin probe H1 to form a 3' blunt end. ExoIII specifically acts on the 3' blunt end of the double-stranded DNA and gradually catalyzes the degradation of one chain in the double-stranded DNA along the 3'→5' direction, causing the hairpin probe to disintegrate, releasing D1 and RP (5'-ATT GCG ATT TCA TAG TGG ATC TTC TGT AC-3'). The D1 chain proceeds to the next cycle, while the RP participates in downstream signal transduction. The large number of RP sequences generated upstream can form base complementary pairing with the sticky end of the B2 sequence in the probe BG that has been pre-assembled through DNA self-assembly, forming the B2-RP probe, competing the B2 sequence off the probe BG. At this time, the middle of the probe BG is in a single-stranded flexible state, and G4-a and G4-b are in the K + With the assistance of the probe, a complete intermolecular DNA G-Quadruplex structure can be formed. The ThT molecules are embedded in the DNA G-Quadruplex structure and aggregate due to the restriction of their intermolecular motion, thereby generating a strong fluorescence signal and realizing the specific amplification detection of Kana. When there is no RP sequence in the system, the split G tetramer sequences G4-a and G4-b in the probe BG cannot approach each other due to the rigid structure formed by the complementary intermediate sequences of B2 and B1, and cannot form a complete intermolecular DNA G tetramer (G-Quadruplex), so no signal response can be performed. The present invention can realize label-free amplification detection of different antibiotics by replacing different antibiotic Aptamer sequences.

[0031] The sensitivity and specificity of the probe system of this solution are reflected in:

[0032] (1) High sensitivity: The specific recognition of Aptamer and the efficient hydrolysis of nuclease III are utilized to realize the recycling of D1 probe, providing more starting sequences RP for downstream signal output reactions, thereby improving detection sensitivity and reaction efficiency; using split-G-Quadruplex as a specific chimeric receptor for ThT signal molecules, a split-G-Quadruplex-ThT signal "lighting up" model is constructed, which can significantly reduce the background signal and improve the detection signal-to-noise ratio.

[0033] (2) High specificity: Aptamer is obtained by screening from a random oligonucleotide library based on the systematic evolution of ligands by exponential enrichment (SELEX) technology, and has high specificity and affinity for the target substance; Nuclease III has a specific hydrolysis effect on the 3' hydroxyl terminal DNA chain of the DNA double-strand, and specifically cuts along the 3'→5' direction, thereby releasing the D1 probe for recycling, and at the same time releasing the RP probe to initiate downstream signal transduction. This process can only be carried out in the presence of the target Kana. The released RP causes the probe BG to undergo a toehold-mediated chain displacement reaction and form a B2-RP probe, further promoting the cleavage probe G4-a and G4-b sequences to approach each other, forming a complete intermolecular G tetramer structure, preparing for the signal "lighting up"; the intermolecular G tetramer structure can specifically bind to the signal molecule ThT, and specifically "lights up" the fluorescent signal by restricting the intermolecular movement of ThT, thereby achieving specific and sensitive detection of the target Kana.

[0034] (3) Low cost: ThT is a water-soluble fluorescent dye. After interacting with DNA G-Quadruplex, the fluorescent signal is significantly enhanced. A label-free signal output model (split-G-Quadruplex-ThT) based on G-Quadruplex "lighting up" ThT can be constructed, which can effectively reduce experimental costs.

[0035] (4) Strong versatility: Specific probes D1 and D2 can be designed for different target apatamines. Combined with the split-G-Quadruplex-ThT initiated by the cyclically generated probe RP, highly sensitive and label-free detection of different antibiotics can be achieved. In addition, the design of probes D1 and D2 can also achieve specific detection of other targets or disease markers such as proteins, nucleic acids, small molecules, and ions. This technology system has strong versatility and a wide range of applications, and can effectively solve the problem of complex design of multiple detection reaction systems in conventional technologies.

[0036] (5) The reaction conditions are mild, the operation is simple, and no expensive temperature-variable instruments are required, which effectively solves the limitations of traditional antibiotic detection.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) We innovatively combined aptamer-driven target-specific recognition, exonuclease III-specific hydrolysis-driven probe recycling, DNA toehold-mediated RP-probe BG-specific competitive reaction, and split-G-Quadruplex-ThT signal "lighting" mechanism to construct a low-background, label-free signal amplification system for the precise detection of the target Kana.

[0039] (2) The probe system of this scheme has the characteristics of high sensitivity, strong specificity, and simple operation, and still maintains good performance in actual samples.

[0040] (3) In addition, by designing the Aptamer probe D2, specific detection of different targets (including other antibiotics, proteins, nucleic acids, small molecules, ions, etc.) can be achieved. The establishment of this technology platform provides a new method for the development of rapid detection kits for disease biomarkers such as small molecules, nucleic acids and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 is the detection mechanism in Example 1;

[0043] Figure 2 Verification of the assembly of D1-D2 and D1-H1 probes in Example 2;

[0044] Figure 3 This is the feasibility analysis of the Exo III drive system cycle in Example 3;

[0045] Figure 4 This is the feasibility analysis of BG probe assembly and RP response in Example 4;

[0046] Figure 5 This is the feasibility analysis of BG probe assembly and RP response in Example 4, where Figure 5 (A) Analysis of the binding specificity between G tetramer and ThT. Figure 5 (B) Feasibility analysis of BG probe and RP response;

[0047] Figure 6 The fluorescence intensity of Kana at different concentrations was detected in Example 5;

[0048] Figure 7 is the linear regression equation of fluorescence intensity in Example 5. DETAILED DESCRIPTION

[0049] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0050] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0051] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0052] The probes involved in the present invention were purchased from Shanghai Sangon Biotechnology Co., Ltd., as shown in Table 1:

[0053]

[0054] Example 1.

[0055] G-tetramer-responsive aptamer-driven cyclic detection of kanamycin DNA probe system:

[0056] (1) DNA probe pretreatment: Prepare lyophilized powders of synthesized probes D1, D2, H1, B1, B2, G4-a, and G4-b into 100 μM stock solutions using DEPC water and store at 4°C for later use.

[0057] (2) Construction of D1 / D2 probes: Probes D1 and D2 were added to a pH 7.4 PBS buffer at a molar ratio of 1:1, annealed at 95°C for 5 min, and then slowly cooled to allow them to self-assemble into D1 / D2 double-stranded probes through base complementary pairing. The probes were then stored in a refrigerator at 4°C for future use.

[0058] (3) Annealing of hairpin probe H1: Anneal the hairpin probe H1 at 95 °C for 5 min and then slowly cool it to allow it to self-assemble into a hairpin structure through base complementary pairing. Store it in a refrigerator at 4 °C for later use.

[0059] (4) Construction of probe BG: Probe B1, probe B2, probe G4-a, and probe G4-b were added to a pH 7.4 PBS buffer at a molar ratio of 1:1:1:1. After annealing at 95 °C for 5 min, the mixture was slowly cooled to allow the probes to self-assemble into probe BG through base complementary pairing. The probes were then stored in a refrigerator at 4 °C for future use.

[0060] (5) Upstream target activation: The sample to be tested is incubated with 1 μM D1 / D2 double-stranded probe and incubated at a constant temperature of 37°C for 30 min. If the sample to be tested is the target Kana, Kana specifically recognizes the aptamer sequence D2 and releases D1 due to the change in the probe conformation. D1 then binds to the hairpin probe H1 through base complementary pairing to form a double-stranded DNA probe. Subsequently, under the action of nuclease III, the double-stranded DNA is specifically cut along the 3'→5' direction, thereby releasing the D1 probe for recycling and simultaneously releasing the RP probe to initiate downstream signal transduction. The feasibility of target activation is verified by electrophoresis and fluorescence experiments.

[0061] (6) Verification of downstream competitive reaction: The probe RP generated by S5 was incubated with 1 μM probe BG constructed by annealing S4, and the B2-RP probe was formed through a toehold-mediated specific chain displacement reaction, thereby bringing the split G4-a and G4-b probes in probe BG closer to each other. + In the presence of 5-nitro-1, DNA tetramer structures can be formed for signal activation; incubate at 37°C for 30 min.

[0062] (7) Signal activation and fluorescence detection of G-Quadruplex-ThT: 40 μM ThT was added to the above system and reacted in PBS buffer (pH 7.4, 50 mM KCl) at 37 °C for 30 min. The fluorescence spectrum of the probe BG after the reaction with RP was collected using an RF-6000 fluorescence spectrophotometer. The excitation wavelength Ex = 450 nm, the emission wavelength Em = 465-600 nm, and the excitation and emission slit widths were set to 5 nm and 5 nm, respectively.

[0063] The detection principle is as follows Figure 1 shown.

[0064] Example 2.

[0065] D1-D2, D1-H1 probe assembly verification:

[0066] The assembly process of D1-D2 and D1-H1 probes was investigated by 12% polyacrylamide gel electrophoresis (PAGE). Figure 2As shown, lane M is the DNA marker, lane 1 is the D1 chain, lane 2 is the D2 chain, lane 5 is D1+D2, and lane 6 is D1+H1. Compared with other control groups, the results demonstrate that D1 and D2 can assemble to form a stable double-stranded probe structure. Furthermore, co-incubation of D1 and H1 also forms a stable double-stranded structure. Notably, no new migrating band was detected in the D2-H1 co-incubation group (lane 7), and its position completely overlapped with the monomeric probe (lane 3), indicating a lack of specific binding between the two. Furthermore, control experiments (lanes 8 / 9) confirmed that the RP chain (lane 4) did not interact with the D1 / D2 chains, eliminating potential interference with the circulation system.

[0067] Example 3.

[0068] Feasibility analysis of Exo III drive system cycle:

[0069] 12% PAGE was used to analyze the feasibility of target recognition and Exo III-driven system circulation, such as Figure 3 As shown, lane 4 shows the D1-D2 probe in the absence of target Kana, while lane 5 shows the result of co-incubation with target Kana and D1-D2. Compared to lane 4 without Kana, the D1-D2 structure band is weaker. This suggests that Kana specifically recognizes and pairs with D2, competing to form D2-Kana, resulting in a decrease in the intensity of the D1-D2 complex band. Notably, no significant band shift was observed in lane 6, where D1-D2 was co-incubated with H1. However, after the introduction of Kana into this system, a new band appeared in lane 7, presumably indicating the release of the D2 chain. Upon further addition of Exo III, a new, faster-migrating band replaced all existing bands, confirming the enzyme's specific cleavage activity against double-stranded DNA. These results indicate that only specific recognition by target Kana can release D1 and bind to H1, and their hybridization further triggers the Exo III-catalyzed reaction. The newly appearing low-molecular-weight bands in lanes 8 and 9 correspond to the H1 digestion product, RP.

[0070] Example 4.

[0071] BG probe assembly and RP response feasibility analysis:

[0072] First, 12% PAGE was used to investigate the stepwise assembly process of probe BG and the feasibility of the chain displacement reaction in the presence of RP, e.g. Figure 4As shown, lane M is a DNA marker, lane 1 is the G4-a chain, lane 2 is the B2 chain, lane 3 is the B1 chain, lane 6 is G4-a+B1+B2, and lane 7 is probe BG (G4-a+B1+B2+G4-b). With the addition of complementary DNA sequences, the PG probe can be successfully assembled. Lane 8 shows the assembly structure of probe BG added to the RP probe (G4-a+). This band corresponds to the result in lane 6. The electrophoresis results show that the probe BG designed in this scheme can be successfully assembled and can achieve a competitive response with RP.

[0073] Subsequently, a fluorescence spectrophotometer was used to investigate the significantly enhanced fluorescence property of ThT molecules after interaction with split-G-Quadruplex DNA G tetramers (split-G-Quadruplex). A label-free fluorescence signal output mode (split-G-Quadruplex-ThT) in which split-G-Quadruplex "lits up" ThT was constructed. The binding specificity of G tetramers to ThT was verified using an RF-6000 fluorescence spectrophotometer. DNA sequences such as R-21 (random sequence, SEQ ID NO. 8), G-21 (SEQ ID NO. 9), A-21 (SEQID NO. 10), T-21 (SEQ ID NO. 11), and C-21 (SEQ ID NO. 12) were selected as controls. The results are as follows: Figure 5 As shown in (A), ThT molecules have almost no fluorescence signal in solution. When they bind to the G tetramer sequence, their fluorescence signal is significantly enhanced. At the same time, when the B2 sequence is missing in the probe BG (G4-a+B1+G4-b), the lack of rigid structural support of the double-stranded probe allows G4-a and G4-b to approach each other and form a complex structure at K. + When present, it can form an intermolecular DNA G tetramer structure, providing a binding site for the embedding of ThT, thereby achieving a significant signal increase, indicating that it has G4 binding specificity. The feasibility of the response of probe BG to RP was further investigated, and the results were as follows Figure 5 As shown in (B), in the absence of probe BG, ThT only shows a weak fluorescence signal; however, when RP is present, the fluorescence signal is significantly enhanced after co-incubation of probe BG and ThT molecules (G4-a+B1+B2+G4-b+RP), indicating that the system has RP response feasibility and is expected to be used for label-free and specific detection of the related target Kana.

[0074] Example 5.

[0075] Analysis of probe BG's response performance to targets:

[0076] In order to evaluate the response ability of probe BG to the target Kana molecule in detail, the fluorescence response behavior of the system was studied with different concentrations of Kana (2 nM, 5 nM, 8 nM, 10 nM, 13 nM, 16 nM). Figure 6 As shown in Figure 2, with the increase of Kana concentration, the ThT fluorescence signal gradually increased, and there was a certain linear relationship in the range of 2-16 nM. Figure 7 As shown, the linear regression equation y = 346.77x + 9686 was obtained, with a linear correlation coefficient R² = 0.9343 and a limit of detection (LOD) of 0.78 nM. The results showed that the ExoⅢ-initiated cycle synergistically combined with the split-G-Quadruplex-ThT signal amplification mechanism can achieve highly sensitive detection of Kana.

[0077] The above is only a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiment. For those skilled in the art, improvements and modifications obtained without departing from the technical concept of the present invention should also be considered as the scope of protection of the present invention.

Claims

1. A DNA probe system for detecting kanamycin based on an aptamer-driven cyclic detection method based on G-tetramer response, characterized in that: The system includes probe D1, probe D2, hairpin probe H1, probe BG, and exonuclease III. Probes D1 and D2 self-assemble into a double-stranded probe through base complementary pairing. Probe BG is self-assembled by probes B1, B2, G4-a, and G4-b through base complementary pairing. In the presence of kanamycin, kanamycin is specifically recognized by probe D1, and detection of kanamycin is achieved through multiple signal amplification. The sequence of the probe D1 is shown in SEQ ID NO. 1; The sequence of the probe D2 is shown in SEQ ID NO. 2; The sequence of the hairpin probe H1 is shown in SEQ ID NO. 3; The sequence of the probe B1 is shown in SEQ ID NO. 4; The sequence of the probe B2 is shown in SEQ ID NO. 5; The sequence of the probe G4-a is shown in SEQ ID NO. 6; The sequence of the probe G4-b is shown in SEQ ID NO.

7.

2. A method for detecting kanamycin using the DNA probe system according to claim 1 for non-diagnostic purposes, characterized in that: The following steps are involved: S1. DNA probe pretreatment: Prepare lyophilized powders of synthesized probes D1, D2, H1, B1, B2, G4-a, and G4-b into 100 µM stock solutions with DEPC water and store at 4°C until use. Construction of double-stranded probes S2 and D1 / D2: Probes D1 and D2 were added to a PBS buffer at pH 7.4 at a molar ratio of 1:

1. The probes were annealed at 95°C for 5 min and then slowly cooled to allow them to self-assemble into double-stranded probes D1 / D2 through complementary base pairing. The probes were then stored in a refrigerator at 4°C until use. S3. Annealing of hairpin probe H1: Anneal the hairpin probe H1 at 95°C for 5 min and then slowly cool it to allow it to self-assemble into a hairpin structure through base complementary pairing. Store it in a refrigerator at 4°C until use. S4. Construction of probe BG: Probe B1, probe B2, probe G4-a, and probe G4-b were added to PBS buffer at pH 7.4 at a molar ratio of 1:1:1:

1. The mixture was annealed at 95°C for 5 min and then slowly cooled to allow the probes to self-assemble into BG probes through complementary base pairing. The probes were then stored in a refrigerator at 4°C until use. S5. Upstream target activation: The sample to be tested is incubated with 1 µM D1 / D2 double-stranded probe and hairpin probe H1, and exonuclease III is added to release probe D1 for recycling; S6, downstream competitive reaction: 1 μM probe BG constructed by annealing in S4 was added to the system prepared in S5 and incubated together. K + , forming a DNA tetramer structure for signal activation; Signal activation and fluorescence detection of S7, G tetramer-thioflavin T: 40 μM thioflavin T was added to the system prepared by S6, and the reaction was carried out at 37°C in PBS buffer for 30 min. The fluorescence spectrum was collected using an RF-6000 fluorescence spectrophotometer.

3. The method according to claim 2, characterized in that The incubation time in step S5 is 30 minutes, and the incubation temperature is 37°C.

4. The method according to claim 2, characterized in that The incubation time in step S6 is 30 minutes, and the incubation temperature is 37°C.

5. The method according to claim 2, characterized in that The pH of the PBS buffer in step S7 is 7.4 and includes 50 mM KCl.

6. The method according to claim 2, characterized in that In step S7, the fluorescence spectrophotometer is set to an excitation wavelength Ex=450 nm, an emission wavelength Em=465-600 nm, and the excitation and emission slit widths are set to 5 nm and 5 nm, respectively.

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