Rapid drug sensitivity detection method based on conformation conversion generated by DNA nanoflowers

Abstract

The invention discloses a rapid drug sensitivity detection method based on conformation conversion generated by DNA nanoflowers, and belongs to the technical field of drug sensitivity detection. Comprising the following steps: (1) constructing a DNA nanoflower structure based on sequences of G4 and an aptamer; and (2) mixing a to-be-detected sample with the DNA nanoflower, after a detection target in the to-be-detected sample is combined with the aptamer, causing internal conformation of the DNA nanoflower to be converted to form a G4 polymer, then forming a compound by the G4 polymer and heme, and then completing rapid detection through a catalyzed chromogenic reaction. The functionalized DNA nanoflower with a controllable structure is synthesized by virtue of a rolling circle amplification technology. After the aptamer is combined with ATP, the structural conformation of the DNA nanoflower is induced to be rearranged, and peroxidase-like activity and signal output are enhanced. Therefore, the sensitivity of bacteria to antibacterial agents can be quickly reflected under the condition that the bacteria do not need to be cultured to the logarithmic phase.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug sensitivity detection, and particularly relates to a rapid drug sensitivity detection method based on DNA nanoflower conformational transition. BACKGROUND

[0002] Bacterial infection has long affected human health, and antibacterial drugs are an effective treatment. However, due to the lack of rapid and accurate drug sensitivity results, empirical treatment or broad-spectrum antibiotics are the first choice for clinicians. In this context, the inevitable overuse or misuse of antibiotics worldwide has accelerated the generation and spread of antibiotic resistance. The key to rational drug use and control of drug resistance is to conduct an antibacterial drug susceptibility test (AST) on pathogenic bacteria, with the aim of ensuring that clinicians give patients appropriate antibacterial drug treatment. The basic principle of AST is to detect the growth of pathogenic bacteria in a specific concentration of antibacterial drugs. The currently used AST methods and some drug resistance mechanism detection methods have standardized guidelines, but the drug sensitivity detection time in the standardized guidelines usually takes 16-20 hours. Although laboratory automation has made significant progress in improving standardization, throughput, and efficiency, the lengthy processing time of more than ten hours is still a bottleneck for making timely and reasonable treatment decisions.

[0003] Currently, rapid drug sensitivity detection mainly includes gene detection and phenotype detection. The limitations of gene detection are influenced by the detection system, gene copy number, and expression differences. Molecular drug sensitivity results can only speculate the true drug sensitivity results, cannot completely correspond to the gold standard of bacterial drug sensitivity, and cannot detect unknown or atypical drug resistance mechanisms. Rapid phenotypic drug sensitivity, which is consistent with the principle of traditional phenotypic drug sensitivity, is an important development direction of drug sensitivity detection. In terms of detection methods, rapid phenotypic drug sensitivity changes from traditional visual observation to sensitive detection of pathogen appearance, movement, or metabolism, thereby shortening the detection time. Currently, rapid phenotypic drug sensitivity detection methods are diverse, including fluorescence analysis, electrochemical sensing, chemiluminescence, Raman spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and machine learning technology. The total detection time of these methods is about 1-4 hours, and the detection results have certain comparability with the gold standard. However, the development of new rapid drug sensitivity platforms is still limited by the application limitations of markers, complex material components and poor biocompatibility, and tedious and time-consuming operation steps, and there is an urgent need to develop new phenotypic drug sensitivity technologies that are accurate, rapid, and widely applicable.

[0004] In the long-term focus on rapid phenotypic drug sensitivity detection, the key to improving the speed and accuracy of bacterial drug sensitivity analysis lies in the development of a more universal biological sensing recognition mode. This mode is composed of three modules: signal recognition (new target), signal amplification (high sensitivity), and signal transduction (easy recognition).

[0005] (1) Signal recognition (new target): The ideal drug susceptibility detection marker, the primary condition is to have universality, that is, expressed in various types of bacteria. At present, a variety of widely existing markers have been reported, including mannose-binding lectin, antibacterial peptide, alkaline phosphatase, antibody, bacteriophage, nucleic acid, aptamer, peptidoglycan binding protein, etc. Among them, there are not a few markers that can be recognized by a wide range of bacteria, but most of them are limited by heterogeneity, instability, high cost, etc. Adenosine triphosphate (ATP). ATP is composed of three subunits, adenine base, five-carbon ribose and triphosphate, which is an indicator of tricarboxylic acid cycle energy metabolism in bacterial cells, and is widely present in prokaryotic and eukaryotic cells. Its short biological half-life is particularly suitable as a marker for drug susceptibility / sensitivity. When bacterial growth is inhibited or killed, the ATP content in the bacteria immediately decreases or disappears, and its content is linearly related to the concentration of viable bacteria. Therefore, the relationship between ATP and drug sensitivity is studied and applied to the broad-spectrum quantitative detection of bacterial drug susceptibility.

[0006] (2) Signal amplification (high sensitivity): Through aptamers, the detection of ATP can be converted into the detection of nucleic acids, and nucleic acid biosensors (NABs) can integrate nucleic acid amplification, significantly improving the universality of this method. Isothermal nucleic acid amplification (ITA) strategy is efficient, among which rolling circle amplification (RCA) has the advantages of simple design and process (only one enzyme and one primer), and is widely used in biosensors. However, integrating RCA into biosensors may prolong the analysis process, so the research group plans to introduce new DNA nanomaterials, such as hydrogel, high-performance scaffolds and DNA nanoflower (DNF), etc., to effectively shorten the turnaround time and improve the recognition efficiency.

[0007] (3) Signal transduction (easy to identify): In the signal output mode, the color change caused by enzymatic reaction is the easiest to achieve clinical application. The biological sensor with visual colorimetric detection has the advantages of rapidity, simplicity, economy and no need for expensive instruments. Nucleic acid sequences have abundant guanine at specific positions, which can form a four-strand helix structure-G-quadruplex (G4) through strong hydrogen bond interaction. G4 has flexible binding ability and is an important part of the biological sensor. G4 can selectively interact with hemoglobin to form a G4-hematin complex, and the catalytic activity of the peroxidase of the complex can be evaluated by the substrate. The complex catalyzes in the presence of hydrogen peroxide (H2O2), and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate shows a clear color change.

[0008] However, it still has certain defects. For example, the stability of the hydrogel may be affected by environmental factors such as pH, temperature and ionic strength, which may affect its performance in complex biological environments. The preparation process of high-performance scaffolds is relatively complex and the cost is high, which limits its large-scale application. The traditional preparation method of DNA nanoflower has problems such as complex structure design and the need for low-concentration organic dye, and further optimization is still needed in the aspect of multifunctional integration. SUMMARY

[0009] In view of the above problems in the prior art, the present application provides a rapid drug sensitivity detection method based on DNA nanoflower conformational transition. The present application focuses on the energy metabolism indicator ATP in bacterial cells, which is used as the recognition target of rapid phenotypic drug sensitivity biosensing. When the bacteria grow or die, the ATP content in the bacteria immediately decreases or disappears, and its content is linearly related to the concentration of live bacteria. Using the programmability of DNA, a functionalized DNA nanoflower linking ATP aptamer and G4 sequence is designed and synthesized by rolling circle amplification technology. When ATP binds to the aptamer, it will trigger the conformational change of the DNA nanoflower, and the G4 stacks and selectively interacts with hematin to form a G4-hematin complex. The complex catalyzes in the presence of H2O2, and TMB as a substrate shows a clear color change corresponding to the content of ATP, and a rapid, accurate and easy-to-identify phenotypic drug sensitivity detection platform is constructed.

[0010] The application realizes multifunctional integration and rapid and sensitive detection of targets by constructing a DNA nanoflower structure through RCA, taking the DNA nanoflower structure as a recognition, signal transduction and signal enhancement component. The RCA is efficiently amplified, and nucleic acid chains are continuously extended and stacked to prepare a highly ordered DNA nanoflower structure, thereby effectively improving the recognition efficiency of the system to ATP. The aptamer sequence and the G4 sequence are spaced apart from each other, and based on the particularity of the ATP aptamer sequence (the head and tail sequences are complementary after being combined with the target), the G4 monomer is automatically pulled closer when the target exists, and the G4 multimer is formed. The G4 multimer has the advantages of high density, good controllability and stable structure, and can accommodate ligands (hematin) through end, lateral stacking or adjacent embedding. Therefore, when ATP exists, the internal conformation of the DNA nanoflower changes, then the G4 combines with hematin to form a G4-hematin complex, which has peroxidase-like catalytic activity, thereby realizing signal transduction. Meanwhile, the stacking of multiple G4 sequences leads to the stacking of multiple G4-hematin complexes, thereby having higher catalytic activity and realizing signal amplification and enhancement.

[0011] To achieve the above object, the technical scheme adopted by the application to solve its technical problems is:

[0012] A rapid detection method based on conformational transition of DNA nanoflowers, comprising the following steps:

[0013] (1) Constructing a DNA nanoflower structure based on G4 and aptamer sequences;

[0014] (2) Mixing the sample to be detected with the DNA nanoflower, when the detection target in the sample to be detected is combined with the aptamer, the internal conformation of the DNA nanoflower is changed to form a G4 multimer, then the G4 multimer is combined with hematin to form a complex, and then the color development reaction of TMB is completed to realize rapid detection.

[0015] Further, in step (1), the G4 and the aptamer are constructed into the DNA nanoflower through rolling circle amplification.

[0016] Further, the aptamer is an ATP aptamer, and the detection target is ATP.

[0017] Further, the specific process of constructing the DNA nanoflower is:

[0018] S1, annealing

[0019] Mixing a circular template, a primer, a buffer and ultrapure water, heating at 95℃ for 5min, and then naturally cooling at room temperature for 3h; the circular template has ATP aptamer and G4 inserted therein, and the sequence is shown as SEQ ID NO. 3; the sequence of the primer is shown as SEQ ID NO. 4;

[0020] S2, ligation

[0021] In the mixture after annealing, add ligase, incubate at room temperature, and obtain a mixed template of a circular template-primer mixed template;

[0022] S3, rolling circle amplification

[0023] The mixed template obtained in S2 is subjected to rolling circle amplification to prepare the DNA nanoflower.

[0024] Further, the reaction system of the rolling circle amplification comprises: mixed circular template 50 μL, dNTP 20 μL, bovine serum albumin 10 μL, phi29 buffer 10 μL, phi29 polymerase 10 μL, and 200 mM Mg 2+ 10 μL.

[0025] Further, the peroxidase catalytic substrate used in step (2) is hydrogen peroxide, and the substrate of the color reaction is TMB.

[0026] A kit for generating conformational conversion based on the DNA nanoflower comprises the DNA nanoflower.

[0027] The above rapid detection method or kit is used for the use in drug sensitivity detection for non-diagnostic purposes.

[0028] A rapid drug sensitivity detection method for non-diagnostic purposes based on the DNA nanoflower for generating conformational conversion comprises the following steps:

[0029] (1) The content of ATP in the sample to be detected is detected according to the method described above;

[0030] (2) According to the absorbance value, it is determined to be resistant or sensitive.

[0031] Further, if the absorbance is >0.8, it is determined to be resistant to antibiotics; and if the absorbance is <0.55, it is determined to be sensitive to antibiotics.

[0032] The beneficial effects of the present application are:

[0033] The present application synthesizes a functionalized DNA nanoflower with controllable structure by means of rolling circle amplification technology, reflects the activity of bacteria through ATP concentration, and is applied to drug sensitivity detection. When the aptamer is combined with ATP, the structure conformation of the DNA nanoflower is rearranged, the peroxidase-like activity is enhanced, and the signal output is enhanced. Therefore, the sensitivity of bacteria to antibacterial drugs can be quickly reflected without culturing the bacteria to the logarithmic phase.

[0034] The detection method constructed by the present application has simple process flow, short response time, strong visualized results, good universality and expandability, and is helpful for realizing efficient and accurate phenotypic drug sensitivity analysis in clinic, providing technical support for individualized drug use and rational use of antibiotics, and being suitable for hospital clinic, drug development and drug resistance monitoring scenes. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure for the effect of DNA nanoflower conformation change on peroxidase-like activity detection results;

[0036] Figure 2 Figure for the absorbance change of two standard sensitive strains based on ATP and DNA nanoflower conformation change with bacterial concentration without antibiotics; A is Escherichia coli ATCC 25922 peak shape figure; B is Escherichia coli ATCC 25922 concentration-dependent curve; C is Escherichia coli ATCC 25922 standard curve figure; D is Klebsiella pneumoniae ATCC 13883 peak shape figure; E is Klebsiella pneumoniae ATCC 13883 concentration-dependent curve; F is Klebsiella pneumoniae ATCC 13883 standard curve figure;

[0037] Figure 3 Figure for the absorbance change of two standard drug-resistant strains based on ATP and DNA nanoflower conformation change with bacterial concentration without antibiotics; A is Klebsiella pneumoniae ATCC BAA1705 peak shape figure; B is Klebsiella pneumoniae ATCC BAA1705 concentration-dependent curve; C is Klebsiella pneumoniae ATCC BAA1705 standard curve figure; D is Klebsiella pneumoniae ATCC BAA1706 peak shape figure; E is Klebsiella pneumoniae ATCC BAA1706 concentration-dependent curve; F is Klebsiella pneumoniae ATCC BAA1706 standard curve figure;

[0038] Figure 4 Figure for the absorbance at 652 nm of sensitive strains (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883) and drug-resistant strains (clinical strain 1, clinical strain 2) before and after exposure to different antibiotics (piperacillin, meropenem, ciprofloxacin, amikacin). DETAILED DESCRIPTION

[0039] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0040] Example 1 Construction of detection system based on functionalized DNA nanoflower conformation change

[0041] 1. Construction of functional DNA nanoflower

[0042] (1) Sequence preparation

[0043] Suitable G4 sequences and ATP aptamer sequences were screened. Based on the rolling circle amplification method, corresponding primer and template sequences were designed (as shown in Table 1), and the template and primer sequences were diluted to a working concentration of 10 μM using ultrapure water.

[0044] Table 1 Sequence

[0045]

[0046]

[0047] (2) Preparation of annealing reaction system: Add the reagents in Table 2 to 1.5 mL EP tubes respectively.

[0048] Table 2 Annealing reagents

[0049]

[0050] (3) Annealing: After vortexing and mixing thoroughly, heat at 95°C for 5 minutes, and then cool naturally at room temperature for about 3 hours.

[0051] (4) Ligation reaction: Add 3 μL of T4 DNA ligase (4 × 10⁻⁶) to the annealed mixture. 5 Mix thoroughly with a pipette tip or by gentle vortexing (U / mL).

[0052] (5) Incubate the mixture at room temperature for 4 hours to obtain a circular template-primer mixture.

[0053] (6) Preparation of rolling circle amplification reaction system: Add the reagents in Table 3 to a 1.5 mL EP tube on ice and mix well with the tip of a pipette.

[0054] Table 3. DNA Nanoflower Self-Assembly Reagents

[0055]

[0056] (7) Amplification reaction: Seal with sealing film and incubate at 30°C for more than 6 hours.

[0057] (8) Enzyme inactivation treatment: The reaction system was inactivated at 75℃ for 10 min to end the reaction.

[0058] (9) Product purification: Centrifuge the product at 14000g and 20℃ for 10min.

[0059] (10) Resuspension and washing: Remove the supernatant with a pipette, add 100 μL DPBS, mix gently, and resuspend to obtain the DNA nanoflower working solution. Repeat the supernatant removal operation twice.

[0060] (11) Preservation: The final DNA nanoflower working solution was stored at -20 °C for later use.

[0061] 2. Preparation of bacterial drug reaction solution

[0062] (1) Preparation of bacterial suspension: A single colony of target bacteria was inoculated into 5 mL of normal saline, vortexed to mix, and adjusted to 0.5 McFarland units. 100 μL of the bacterial solution was added to 4.9 mL of MH broth, and then 167 μL was added to 5 mL of MH broth to obtain a bacterial suspension of 1 x 10 5 CFU / mL for later use.

[0063] (2) Preparation of antibiotic working solution: The required antibiotic working solution was prepared using MH broth, and the specific concentrations were as follows: piperacillin 16 μg / mL, amikacin 8 μg / mL, ciprofloxacin 0.5 μg / mL, and meropenem 2 μg / mL. The antibiotics were used fresh to prevent hydrolysis.

[0064] (3) Co-incubation of bacteria and drugs: 0.5 mL of the test bacterial solution was thoroughly mixed with 0.5 mL of the corresponding concentration of antibiotic working solution, and then incubated at 37 °C for 60 min. After incubation, the supernatant was discarded by centrifugation at 3500 rpm for 5 min, and the bacterial precipitate was retained.

[0065] (4) Lysis step: The bacterial precipitate was washed with Tris-HCl buffer and resuspended in 500 μL of lysis solution containing 0.5 mg / mL lysozyme and 1 mM EDTA. The cells were lysed at 37 °C for 10 min to promote cell lysis. After lysis, 0.1% Triton X-100 was added, and the mixture was gently mixed at room temperature. Then, the mixture was centrifuged at 12000 g for 3 min, and the supernatant was collected as the bacterial drug reaction solution for subsequent detection.

[0066] Pre-cooled Tris-HCl buffer (10 mM, pH 7.4) was used to reduce the leakage of intracellular ATP. The lysozyme and EDTA were prepared fresh to prevent the degradation of lysozyme activity. After adding Triton X-100, the mixture was gently mixed to avoid DNA fragmentation caused by vigorous shaking.

[0067] 3. Colorimetric detection

[0068] The above-mentioned bacterial drug reaction solution was added to the DNA nanoflower reaction system. After the aptamer recognized ATP, it induced the change of G4 conformation to form a multimer, which combined with hematin to form a G4-hematin complex. This complex has peroxidase-like activity and catalyzes the TMB color reaction in the presence of hydrogen peroxide. Specifically:

[0069] Take 50 μL bacteria drug reaction solution, add 10 μL 300 μM hematin, 20 μL DNA nanoflower working solution, incubate for 20 min, finally add 100 μL pH 4.0 0.1 mol / L acetic acid buffer, 10 μL 100 mM H2O2, 10 μL 20 mM TMB, avoid light reaction for 10 min. The color development result can be quantitatively analyzed by using ultraviolet spectrophotometer at 652 nm wavelength.

[0070] 4. Determination and analysis of detection results:

[0071] If the absorbance is greater than 0.8, it is judged as drug-resistant, and if the absorbance is less than 0.55, it is judged as sensitive. If the bacteria are drug-resistant, the degree of inhibition of bacterial reproduction in the presence of corresponding concentration of antibiotic is low, and the ATP metabolic activity is active. When the bacteria are lysed, the released ATP content is high, the formed complex is more, and the catalysis of H2O2 and TMB reaction is more intense, and the absorbance is higher. On the contrary, if the bacteria are sensitive to the antibiotic, the bacterial reproduction is inhibited, the ATP concentration is low, and the absorbance is low. The experimental results show that the whole process from bacterial inoculation, incubation to ATP detection can be completed in about 2 hours. The results are highly consistent with the traditional 24-hour drug sensitivity test, which verifies the accuracy and clinical feasibility of the method.

[0072] Then the influence of the DNA nanoflower conformation change on the peroxidase-like activity is detected, and the results are shown in Figure 1 .

[0073] As shown in Figure 1 , by comparing the absorbance of DNA nanoflower+ATP+hematin+TMB+H2O2 and DNA nanoflower+hematin+TMB+H2O2, it can be seen that after introducing ATP, the peroxidase-like activity of the domain in the DNA nanoflower is significantly enhanced. This further proves that after introducing ATP, the conformation changes, successfully integrates multiple G4-hematin complexes, and enhances the peroxidase-like activity. Therefore, it has higher catalytic activity, so as to achieve the effect of signal amplification and enhancement.

[0074] Example 2: Differentiation of different bacterial concentrations based on ATP-specific functionalized DNA nanoflower conformation change

[0075] To prove that the conformation of the functionalized DNA nanoflower can change with the change of the ATP content of the bacteria, the present application selects four model strains (two standard sensitive strains: Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 13883; two standard drug-resistant strains: Klebsiella pneumoniae ATCC BAA1705 and ATCC BAA1706) for verification, and the specific process is as follows:

[0076] First, bacterial suspensions (bacterial concentrations of 0, 500, and 10⁻⁶ respectively) were prepared using the same method. 3 10 4 10 5 10 6 10 7 (CFU / mL) 1 mL of bacterial suspension was centrifuged at 3500 rpm for 5 min, the supernatant was discarded, and the bacterial pellet was retained. The pellet was washed with Tris-HCl buffer and resuspended in 500 μL of lysis buffer (containing 0.5 mg / mL lysozyme and 1 mM EDTA). The pellet was incubated at 37°C for 10 min to promote cell lysis. After lysis, Triton X-100 (final concentration 0.1%) was added to aid complete lysis. After mixing at room temperature, the pellet was centrifuged at 12000 g for 3 min, and the supernatant was collected as the bacterial lysis buffer. 50 μL of the bacterial lysis buffer was added to 20 μL of DNA nanoflower working solution and incubated for 20 min. Finally, 100 μL of 0.1 mol / L sodium acetate buffer (pH 4.0), 10 μL of 100 mM H₂O₂, and 10 μL of 20 mM TMB were added sequentially, and the reaction was carried out in the dark for 10 min. The results are shown in the figure. Figure 2 and Figure 3 .

[0077] like Figure 2 , Figure 3 As shown, regardless of whether it is *Escherichia coli* or *Klebsiella pneumoniae*, whether it is a susceptible or drug-resistant strain, the ATP content and absorbance increase with increasing bacterial concentration. Within a certain bacterial concentration range (10... 3 -10 7 The positive correlation between CFU / mL and Log[bacterial concentration] and absorbance demonstrates that it is feasible to distinguish different bacterial concentrations based on ATP-specific functionalized DNA nanoflower conformational changes, and provides an analytical basis for subsequent rapid drug sensitivity analysis.

[0078] Example 3: Application of ATP-specific functionalized DNA nanoflower conformational changes to rapid drug sensitivity detection

[0079] The model strains were clinically isolated fully drug-resistant strains (clinical strain 1 and clinical strain 2) from standard strains (Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 13883). Commonly used clinical antibiotics (piperacillin, meropenem, ciprofloxacin, and amikacin) were used as model drugs. Drug concentrations were determined according to the standardized guidelines CLSI, and their breakpoints were used as the standard. The relationship between susceptible and drug-resistant strains and the ATP results before and after antibiotic incubation was verified. The results are shown in […]. Figure 4 .

[0080] like Figure 4As shown in A and C, the standard sensitive strains have obvious changes in absorbance before and after different antibiotic exposure, and are judged to be sensitive to piperacillin, meropenem, ciprofloxacin and amikacin. Figure 4 As shown in B and D, the whole drug-resistant clinical isolates have no obvious changes in absorbance before and after different antibiotic exposure, and are judged to be resistant to piperacillin, meropenem, ciprofloxacin and amikacin, which is consistent with the results of the conventional KB method. This result further verifies that the rapid drug sensitivity technology based on the conformational change of the functionalized DNA nanoflower specific to ATP can be used for actual clinical drug sensitivity result analysis.

[0081] Finally, it should be pointed out that the above specific embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application is described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A rapid detection method based on DNA nanoflower generating conformational conversion, characterized in that, The method comprises the following steps: (1) constructing a DNA nanoflower structure based on G4 and aptamer sequences; (2) mixing the sample to be tested with the DNA nanoflower, and when the detection target in the sample to be tested binds with the aptamer, the internal conformation of the DNA nanoflower is converted to form a G4 multimer, then the G4 multimer forms a complex with hematin, and finally the rapid detection is completed through catalytic color development reaction. 2.The rapid detection method based on DNA nanoflower generated conformational conversion according to claim 1, wherein, The G4 and aptamer-based sequences in step (1) are constructed by rolling circle amplification. 3.The rapid detection method based on DNA nanoflower generated conformation transition according to claim 1 or 2, characterized in that, The aptamer is an ATP aptamer, and the detection target is ATP. 4.The rapid detection method based on DNA nanoflower generated conformation transition of claim 3, wherein, The specific process of constructing the DNA nanoflower is as follows: S1, annealing Mix the circular template, primer, buffer and ultrapure water, heat at 95℃ for 5 min, and then naturally cool at room temperature for 3 h; the circular template has ATP aptamer and G4 inserted therein, and the sequence is shown as SEQ ID NO. 3; The sequence of the primer is shown as SEQ ID NO. 4; S2, ligation Add ligase to the mixture after annealing, incubate at room temperature, and obtain a mixed template of the circular template-primer mixture; S3, rolling circle amplification Perform rolling circle amplification on the mixed template obtained in S2 to prepare the DNA nanoflower.

5. The rapid detection method based on DNA nanoflower generated conformational conversion according to claim 4, characterized in that, The reaction system of rolling circle amplification includes: mixed circular template 50 μL, dNTP 10 μL, bovine serum albumin 10 μL, phi29 buffer 10 μL, phi29 polymerase 10 μL, and 200 mM Mg 2+ 10 μL. 6.The rapid detection method based on DNA nanoflower generated conformation transition of claim 1, wherein, The peroxidase catalytic substrate used in step (2) is hydrogen peroxide, and the color development reaction substrate is TMB.

7. A kit for generating a conformational shift based on DNA nanoflow, characterized by, The DNA nanoflower as claimed in any one of claims 1 to 6.

8. Use of the rapid detection method as claimed in any one of claims 1 to 6 or the kit as claimed in claim 7 in drug sensitivity detection for non-diagnostic purposes.

9. A rapid drug susceptibility testing method based on DNA nanoflower generated conformational switching for non-diagnostic purposes, characterized by, The method comprises the following steps: (1) detecting the content of ATP in the sample to be tested according to the method described in claim 1; (2) determining whether it is drug-resistant or sensitive according to the absorbance value.

10. The rapid antimicrobial susceptibility testing method of claim 9, wherein, If the absorbance is > 0.8, it is determined to be resistant to antibiotics; and if the absorbance is < 0.55, it is determined to be sensitive to antibiotics.

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