An aptamer sensor and preparation and application thereof

Abstract

The application relates to an aptamer sensor and preparation and application thereof. The aptamer sensor comprises an aptamer chain, an auxiliary chain, an LH1 chain, an LH2 chain and a buffer system, the LH1 chain is formed by assembling an L chain and a plurality of hairpin probes H1 through base complementary pairing, and the LH2 chain is formed by assembling an L chain and a plurality of hairpin probes H2 through base complementary pairing; the hairpin probe H1 comprises a first hairpin sequence section and an L chain complementary section, the first hairpin sequence section participates in forming a stem loop structure of the hairpin probe H1 through intrachain base complementary pairing, meanwhile, the first hairpin sequence section serves as a core region which is specifically complementary to the auxiliary chain, can be combined with the auxiliary chain and triggers a sticky end-mediated strand displacement reaction, and realizes opening of a hairpin secondary structure; the hairpin probe H2 comprises a second hairpin sequence section and an L chain complementary section, and the first hairpin sequence section and the second hairpin sequence section are specifically complementary to each other.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, and in particular to an aptamer sensor and its preparation and application. Background Technology

[0002] Chloramphenicol (CAP), a highly effective broad-spectrum antibiotic, was once widely used in medical treatment and growth promotion in livestock and poultry farming. However, long-term intake can cause serious health problems such as bone marrow suppression and aplastic anemia. Therefore, the accurate detection of CAP residues in food and medicine is of great practical significance.

[0003] Currently, the main detection methods for CAP include liquid chromatography, electrochemical methods, enzyme-linked immunosorbent assay (ELISA), colorimetry, and fluorescence methods. Among these, fluorescence methods have become one of the mainstream technologies for antibiotic detection due to their advantages such as high sensitivity, low cost, and simple operation. Meanwhile, nucleic acid aptamers, with their high specificity, high affinity, and strong stability, are often used as recognition elements and combined with fluorescence methods to construct aptamer sensors.

[0004] To further improve detection sensitivity, signal amplification techniques are widely used in the construction of aptamer sensors, mainly including enzyme-catalyzed amplification, nanomaterial-assisted amplification, and DNA self-assembly amplification. Enzyme-catalyzed amplification relies on the catalytic activity of enzymes, but suffers from problems such as high enzyme cost and easy inactivation; nanomaterial-assisted amplification techniques (such as gold nanoparticles and quantum dots) require complex material preparation processes; DNA self-assembly amplification techniques (such as CHA and HCR) do not require enzyme participation, thus avoiding the above-mentioned drawbacks.

[0005] However, in traditional CHA technology, the hairpin probes H1 and H2 are in a free-dispersed state, resulting in low local concentrations of reactants. This leads to a slow chain displacement reaction rate and a long detection time (usually more than 3 hours). Freely dispersed hairpin probes are prone to non-specific hybridization, resulting in high background fluorescence signals, which affects the specificity and sensitivity of the detection.

[0006] To address the slow speed of DNA self-assembly reactions, researchers have attempted to increase the local concentration of DNA reactants through spatial localization, such as the L-DCDR / MB-DNSs system and AuNPs to fix DNA strands. However, these methods have drawbacks such as complex design, cumbersome preparation steps, and time consumption, making it difficult to achieve rapid and efficient detection. Summary of the Invention

[0007] The present invention aims to disclose an aptamer sensor and its preparation and application, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide an aptamer sensor. The aptamer sensor includes an aptamer chain, an auxiliary chain, an LH1 chain, an LH2 chain, and a buffer system. The auxiliary chain and the aptamer chain are annealed to form an AP-CAPaptamer double chain. The LH1 chain is formed by assembling an L chain with multiple hairpin probes H1 through complementary base pairing. The LH2 chain is formed by assembling an L chain with multiple hairpin probes H2 through complementary base pairing. The hairpin probe H1 comprises a first hairpin sequence segment and an L-chain complementary segment from the 5' end to the 3' end. The 5' end of the hairpin probe H1 is labeled with a fluorescent group, and a fluorescent quenching group is inserted between the first hairpin sequence segment and the L-chain complementary segment. The first hairpin sequence segment participates in the formation of the stem-loop structure of the hairpin probe H1 through intra-chain base complementary pairing. At the same time, the first hairpin sequence segment, as a core region specifically complementary to the auxiliary chain, can bind to the auxiliary chain and trigger a sticky end-mediated chain substitution reaction to open the hairpin secondary structure. The hairpin probe H2 includes a second hairpin sequence segment and an L-chain complementary segment from the 5' end to the 3' end, wherein the first hairpin sequence segment and the second hairpin sequence segment are specifically complementary.

[0009] The aptamer sensor described in the first aspect of this invention employs a "dispersed-localized" catalytic hairpin assembly (DL-CHA) strategy. This strategy transforms hairpin probes H1 and H2 from a freely dispersed state to a locally concentrated state via the L-chain, significantly increasing the local concentration of DNA reactants and thus significantly improving detection efficiency. The first hairpin sequence segment of hairpin probe H1 has dual functions of stem-loop structure formation and auxiliary strand complementary triggering, eliminating the need for additional specific triggering segments and simplifying probe sequence design. Simultaneously, the stem-loop structure formed through intra-strand base complementarity ensures that the 5' fluorophore and the intermediate quenching group are spatially adjacent, achieving fluorescence quenching and guaranteeing low initial background fluorescence for the sensor. The specific complementary pairing of the first and second hairpin sequence segments provides the structural basis for catalytic hairpin assembly cyclic amplification, increasing detection sensitivity to 2.4 × 10⁻⁶ to 7.4 × 10⁻⁶. 3 The reaction time to reach equilibrium is reduced from more than 3 hours in the traditional CHA to 2.5 hours.

[0010] In a further embodiment of the first aspect of the present invention, the fluorescent group is selected from FAM, HEX, TET, JOE or FITC; the quenching group is selected from BHQ1 or BHQ2.

[0011] In a further embodiment of the first aspect of the present invention, the L-chain contains at least two probe complementary segments, which specifically complement each other with the L-chain complementary segments. The presence of multiple probe complementary segments on the L-chain significantly increases the probe density on the LH1 or LH2 chain. Simultaneously, through targeted assembly on the L-chain, non-specific hybridization of the hairpin probes is reduced, significantly lowering the background fluorescence signal and improving cyclic amplification efficiency, resulting in a detection limit as low as 6.3 × 10⁻⁶. -4 pmol / L, which is far superior to the traditional CHA sensor (4.3 pmol / L) and other existing detection methods.

[0012] In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the L chain is: 5'-TTGGGTGTGAGAGTGTTGGGTGTGAGAGTGTTGGGTGTGTGAGAGTGTTGGGTGTGTGAGAGTGTTGGGTGTGAGAGTG-3' (SEQ ID No: 3). It contains five repeating probe complementary segments, which can efficiently achieve specific base pairing with the complementary segments of the L chain of the hairpin probes H1 / H2, resulting in high local assembly efficiency and significantly increasing the local effective concentration of the hairpin probes. Furthermore, this sequence is a linear single-stranded structure without complex secondary structures, preventing non-specific binding with other chains and ensuring the specificity of local assembly. Simultaneously, the base composition and length of this sequence are suitable for enzyme-free isothermal reaction systems, remaining stable under 37°C incubation conditions without degradation, further improving the stability and detection repeatability of the sensor.

[0013] In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the hairpin probe H1 is: 5'-ACTCAGCCACCATCAGATGTGTAGAGATGGTGGCTGAG-TCTACAACTTTTCACTCTCACACCCAA-3' (SEQ ID No: 4). The first hairpin sequence segment of this sequence can efficiently form a stable stem-loop structure through intra-strand base complementarity, ensuring the spatial proximity of the fluorescent group and the quenching group, achieving efficient fluorescence quenching and reducing background signal; at the same time, the base sequence of this segment achieves precise and specific complementarity with the auxiliary strand, resulting in high chain substitution reaction triggering efficiency and rapid opening of the hairpin structure to achieve fluorescence signal recovery; and the L-strand complementary segment of this sequence has high pairing efficiency with the probe complementary segment of the L-strand, resulting in good local assembly effect, effectively increasing the local concentration of the probe, accelerating the subsequent amplification reaction, and further improving the detection sensitivity and detection speed of the sensor.

[0014] In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the hairpin probe H2 is: 5'-CTCAGCCACCATCTCTACACATCTGATGGTGGCTGAGATCGAATTTTCACTCTCACACCCAA-3' (SEQ ID No: 5). The second hairpin sequence segment of this sequence can achieve highly efficient and specific complementary pairing with the first hairpin sequence segment, resulting in high binding efficiency for the hairpin assembly reaction. This allows for rapid completion of probe assembly and cyclic release of the auxiliary strand, achieving cyclic amplification of the fluorescence signal and significantly improving the detection sensitivity of the sensor. Simultaneously, the complementary segment of the L chain precisely pairs with the complementary segment of the probe on the L chain, ensuring the structural compatibility of the LH1 and LH2 chains, enabling the catalytic hairpin assembly reaction to proceed in an orderly manner and avoiding non-specific amplification. Furthermore, the base composition of this sequence is compatible with enzyme-free isothermal systems, exhibiting good stability under reaction conditions, further enhancing the detection repeatability and applicability of the sensor for actual sample detection.

[0015] In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the auxiliary strand is: 5'-AGTCGCTCAGCCACCATC-3' (SEQ ID No: 2). Its base sequence is precisely and specifically complementary to the first hairpin sequence segment, which can efficiently trigger the sticky-end-mediated strand substitution reaction. While opening the hairpin structure, it can also achieve its own cyclic release, providing continuous triggering power for multiple rounds of catalytic hairpin assembly, thus achieving efficient amplification of the fluorescence signal. Furthermore, this sequence is a short-chain single-strand structure, which diffuses rapidly in the reaction system, further improving the rate of the strand substitution and amplification reactions and shortening the detection time.

[0016] In a further embodiment of the first aspect of the present invention, the aptamer chain is a chloramphenicol aptamer with the nucleotide sequence: 5'-GATGGTGGCTGAGCGGCTGGCACCCTGTTGAGTGACTTCA-3' (SEQ ID No: 1). This sequence is an aptamer for chloramphenicol and can bind to chloramphenicol molecules with high affinity and high specificity. The AP-CAPaptamer double strand assembled therefrom undergoes a conformational change after binding to chloramphenicol molecules and efficiently releases the auxiliary strand, achieving precise target recognition and signal triggering, avoiding non-specific binding of other antibiotics or impurities, and significantly improving the sensor's specificity for chloramphenicol detection.

[0017] A second aspect of this invention provides a method for detecting chloramphenicol. The detection method includes the following steps: 1) Obtain the aptamer strand with the nucleotide sequence shown in SEQ ID No:1 and the auxiliary strand with the nucleotide sequence shown in SEQ ID No:2, and anneal them to form an AP-CAP aptamer double strand; obtain the hairpin probe H1 with the nucleotide sequence shown in SEQ ID No:4, anneal it to form a hairpin structure, and then incubate it with the L strand with the nucleotide sequence shown in SEQ ID No:3 to form the LH1 strand; obtain the hairpin probe H2 with the nucleotide sequence shown in SEQ ID No:5, anneal it to form a hairpin structure, and then incubate it with the L strand with the nucleotide sequence shown in SEQ ID No:3 to form the LH2 strand; 2) Incubate the AP-CAP aptamer double strand with the sample to be tested to release the auxiliary strand; 3) Add the LH1 chain and the LH2 chain to the reaction system of step 2) to carry out a catalytic hairpin assembly reaction; 4) Detect the fluorescence intensity of the reaction system in step 3), and calculate the chloramphenicol content in the sample by using the linear regression equation obtained from the working curve.

[0018] The detection method is based on strand displacement reaction (TSDR) and a "dispersion-localization" catalytic hairpin assembly (DL-CHA) strategy to construct an enzyme-free nucleic acid isothermal amplification aptamer sensor. When CAP is present in the sample, CAP specifically binds to the CAP-aptamer double strand, causing the release of the auxiliary strand. The released auxiliary strand sequentially opens the hairpin structure of hairpin probe H1 in the LH1 strand and the hairpin structure of hairpin probe H2 in the LH2 strand through the TSDR reaction, completing one round of CHA reaction. As the hairpin structure of hairpin probe H1 is opened, its 5' end fluorescent group separates from the mid-segment quenching group, generating fluorescence. At the same time, the auxiliary strand is released again, entering the next cycle, continuously triggering the assembly of LH1 and LH2 strands to form a ladder-like DNA strand structure, achieving efficient amplification of the fluorescence signal.

[0019] In a further embodiment of the second aspect of the present invention, the preparation method of the AP-CAP aptamer double strand is as follows: 20 μL of 10 μmol / L auxiliary chain and 20 μL of 10 μmol / L aptamer chain are mixed with 210 μL of PBS buffer solution with pH=7.4 and 0.02 mol / L, annealed in a water bath at 95 °C for 5 min, and then naturally cooled to room temperature and stored at 4 °C for later use.

[0020] In a further embodiment of the second aspect of the present invention, the LH1 chain and the LH2 chain are prepared as follows: 20 μL of 50 μmol / L hairpin probe H1 or hairpin probe H2 is added to 960 μL of 0.02 mol / L PBS buffer solution (pH=7.4), annealed in a water bath at 95°C for 5 min, and then naturally cooled to room temperature to form a stable hairpin structure; then, 20 μL of 10 μmol / L L chain is added to the solution containing the hairpin structure, incubated at 37°C for 1 h, and the LH1 chain or LH2 chain is assembled and stored at 4°C for later use.

[0021] This invention provides an improved chloramphenicol detection method with high sensitivity, simple operation, and low cost. It requires no enzyme catalysis or complex nanomaterial preparation, achieving localization and amplification solely through complementary base pairing of DNA strands. The preparation process is simple and the detection cost is low. Validation results show a spiked recovery rate of 98.6%–104.1% and an RSD ≤ 4.7% for actual samples (chloramphenicol eye drops, milk), demonstrating good accuracy and stability, and meeting the practical detection needs of CAP residues in food and pharmaceuticals. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of CAP detection by the aptamer sensor. Figure 2 This is an agarose gel electrophoresis image from Example 3. Detailed Implementation

[0023] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0024] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] The instruments and equipment involved in the embodiments include: a fluorescence spectrometer (to detect fluorescence intensity), an agarose gel electrophoresis apparatus (to verify the reaction process), a pH meter (to adjust the pH value), and a constant temperature water bath (to control the reaction temperature).

[0026] Example 1: Construction of a chloramphenicol aptamer sensor 1) Preparation of AP-CAP aptamer double strands: 20 μL of auxiliary strand (10 μmol / L), 20 μL of chloramphenicol aptamer strand (10 μmol / L) and 210 μL of PBS buffer solution (pH=7.4, 0.02 mol / L) were mixed and annealed in a 95 ℃ water bath for 5 min. After cooling naturally to room temperature, AP-CAP aptamer double strands were formed and stored at 4 ℃ for later use.

[0027] 2) Take 20 μL of hairpin probe H1 (50 μmol / L) and 20 μL of hairpin probe H2 (50 μmol / L) respectively, add 960 μL of PBS buffer solution (0.02 mol / L), anneal in a water bath at 95 ℃ for 5 min, and then cool naturally to room temperature to form a stable hairpin structure; Add 20 μL of L chain (10 μmol / L) to the hairpin probe H1 and hairpin probe H2 solutions that have formed stable hairpin structures, respectively, and incubate at 37 °C for 1 h. The LH1 and LH2 chains are assembled through base complementary pairing and stored at 4 °C for later use.

[0028] 3) Take 10 μL of AP-CAP aptamer double strand (4 μmol / L) and add it to a centrifuge tube, then add 10 μL of CAP standard solution of different concentrations, and incubate at room temperature for 45 min to allow CAP to specifically bind to CAP-aptamer and release the auxiliary strand; 4) Add 40 μL of LH1 and LH2 chain mixture to a centrifuge tube and react in a 37℃ water bath for 2.5 h to trigger the DL-CHA cyclic amplification reaction; 5) Use a fluorescence spectrometer to measure the fluorescence intensity (F) of the reaction solution at 528 nm, and at the same time measure the fluorescence intensity (F0) of the blank group (without CAP), and calculate the change in fluorescence intensity ΔF = F - F0; 6) Plot the working curve with ΔF as the ordinate and the logarithm of CAP concentration as the abscissa, and establish a linear regression equation. F =109.4lg C CAP +1813.7 ( C CAP The unit is pmol / L), and the regression coefficient R is... 2 The value was 0.994, and the limit of detection (LOD) was 6.3 × 10⁻⁶. - 4 pmol / L (3σ / S).

[0029] The detection principle of the chloramphenicol aptamer sensor is as follows: Figure 1 As shown.

[0030] Example 2, Actual Sample Processing The detection performance of the chloramphenicol aptamer sensor was verified using chloramphenicol eye drops and milk as samples, respectively.

[0031] (1) Take commercially available chloramphenicol eye drops and dilute 129.2 μL of the sample to 60 pmol / L according to the concentration indicated on the product label (20.0 mg / 8.0 mL, i.e. 7.74 mmol / L).

[0032] The concentration was 57.9 pmol / L, as determined by the detection method provided in Example 1.

[0033] (2) Take 5.0 mL of milk sample, add 4.0 mL of 1% trichloroacetic acid, mix and sonicate for 20 min, then centrifuge at 10000 rpm for 10 min; take the supernatant and filter it with 0.22 μm membrane filter paper, dilute the filtrate to 10 mL, and use it as the test sample; take 10 μL of the treated sample solution and determine it according to the detection procedure provided in Example 1, and at the same time perform the spike recovery experiment. The results are shown in Table 1.

[0034] Table 1 - Determination and Spike Recovery of Chloramphenicol Aptamer Sensor in Chloramphenicol Eye Drops and Milk Samples

[0035] ND: Indicates not detected.

[0036] Example 3, Gel electrophoresis detection To demonstrate the detection principle of the described ligand sensor, verification was performed using 2% agarose gel electrophoresis: (1) Prepare an agarose gel with a concentration of 2% (w / w); (2) Prepare DNA samples in different lanes, wherein lanes 1 to 8 are AP strand, L strand, hairpin probe H1, hairpin probe H2, LH1 double strand, LH2 double strand, LH1+LH2 double strand and AP-CAP aptamer+CAP+LH1+LH2 strand respectively; (3) Place the prepared agarose gel in a 0.5×TBE (44.5 mmol / L Tris-H3BO3, 1 mmol / L EDTA, pH=8.0) solution, then mix 10 μL of DNA samples from different lanes with 2 μL of 6×glycerol loading buffer, and add 10 μL to the corresponding lane. Run the gel at 101 V for 60 minutes and then take it out to observe the imaging under a UV lamp.

[0037] Test results as follows Figure 2As shown. Compared to the original DNA single strands (lanes 1-4), after the L strand hybridized with hairpin probe H1 or hairpin probe H2, lanes 5 and 6 showed a weakening or even disappearance of the original strand signal, and the formation of new strands with a low electrophoretic migration rate, proving the formation of new LH1 and LH2 strands; only LH1 and LH2 strands were mixed, and no new bands appeared in lane 7 except for the bands of LH1 and LH2 strands, that is, no new strands were formed, indicating that the mixing of LH1 and LH2 strands does not result in a hybridization reaction; however, when the target compound CAP was added, two new bands appeared in lane 8, one of which was a band with a slower migration rate. The addition of the target compound CAP caused AP-CAP to... After the aptamer double helix releases its auxiliary strand, it sequentially opens the LH1 and LH2 chains to form a larger DL-CHA chain with a DNA ladder structure, thus proving the formation of a new substance, DL-CHA. Another band appears almost identically to band 1 in lane 1; this band represents the release of the auxiliary strand due to the specific interaction between the target molecule CAP and its chloramphenicol aptamer chain. Therefore, the gel electrophoresis experiment confirms the occurrence of the chain substitution reaction in the experimental principle.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An aptamer sensor, characterized in that, It includes an aptamer chain, an auxiliary chain, an LH1 chain, an LH2 chain, and a buffer system. The auxiliary chain and the aptamer chain are annealed to form an AP-CAP aptamer double chain. The LH1 chain is formed by assembling an L chain with multiple hairpin probes H1 through complementary base pairing. The LH2 chain is formed by assembling an L chain with multiple hairpin probes H2 through complementary base pairing. The hairpin probe H1 comprises a first hairpin sequence segment and an L-chain complementary segment from the 5' end to the 3' end. The 5' end of the hairpin probe H1 is labeled with a fluorescent group, and a fluorescent quenching group is inserted between the first hairpin sequence segment and the L-chain complementary segment. The first hairpin sequence segment participates in the formation of the stem-loop structure of the hairpin probe H1 through intra-chain base complementary pairing. At the same time, the first hairpin sequence segment, as a core region specifically complementary to the auxiliary chain, can bind to the auxiliary chain and trigger a sticky end-mediated chain substitution reaction to open the hairpin secondary structure. The hairpin probe H2 includes a second hairpin sequence segment and an L-chain complementary segment from the 5' end to the 3' end, wherein the first hairpin sequence segment and the second hairpin sequence segment are specifically complementary.

2. The aptamer sensor according to claim 1, characterized in that, The L-chain contains at least two probe complementary segments, which are specifically complementary to the L-chain complementary segments.

3. The aptamer sensor according to claim 2, characterized in that, The nucleotide sequence of the L chain is shown in SEQ ID No:

3.

4. The aptamer sensor according to claim 3, characterized in that, The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID No:

4.

5. The aptamer sensor according to claim 4, characterized in that, The nucleotide sequence of the hairpin probe H2 is shown in SEQ ID No:

5.

6. The aptamer sensor according to claim 4, characterized in that, The nucleotide sequence of the auxiliary chain is shown in SEQ ID No:

2.

7. The aptamer sensor according to claim 6, characterized in that, The aptamer chain is a chloramphenicol aptamer, and its nucleotide sequence is shown in SEQ ID No:

1.

8. A method for detecting chloramphenicol, characterized in that, Including the following steps: 1) Obtain the aptamer strand with the nucleotide sequence shown in SEQ ID No:1 and the auxiliary strand with the nucleotide sequence shown in SEQ ID No:2, and anneal them to form an AP-CAP aptamer double strand; obtain the hairpin probe H1 with the nucleotide sequence shown in SEQ ID No:4, anneal it to form a hairpin structure, and then incubate it with the L strand with the nucleotide sequence shown in SEQ ID No:3 to form the LH1 strand; obtain the hairpin probe H2 with the nucleotide sequence shown in SEQ ID No:5, anneal it to form a hairpin structure, and then incubate it with the L strand with the nucleotide sequence shown in SEQ ID No:3 to form the LH2 strand; 2) Incubate the AP-CAP aptamer double strand with the sample to be tested to release the auxiliary strand; 3) Add the LH1 chain and the LH2 chain to the reaction system of step 2) to carry out a catalytic hairpin assembly reaction; 4) Detect the fluorescence intensity of the reaction system in step 3), and calculate the chloramphenicol content in the sample by using the linear regression equation obtained from the working curve.

9. The chloramphenicol detection method according to claim 8, characterized in that, The preparation method of the AP-CAP aptamer double strand is as follows: 20 μL of 10 μmol / L auxiliary chain and 20 μL of 10 μmol / L aptamer chain are mixed with 210 μL of 0.02 mol / L PBS buffer solution with pH=7.

4. After annealing in a water bath at 95 ℃ for 5 min, the mixture is naturally cooled to room temperature and stored at 4 ℃ for later use.

10. The chloramphenicol detection method according to claim 8, characterized in that, The LH1 and LH2 chains are prepared as follows: Take 20 μL of 50 μmol / L hairpin probe H1 or hairpin probe H2, add 960 μL of 0.02 mol / L PBS buffer solution (pH=7.4), anneal in a water bath at 95℃ for 5 min, and then cool naturally to room temperature to form a stable hairpin structure; then add 20 μL of 10 μmol / L L chain to the solution containing the hairpin structure, incubate at 37℃ for 1 h, assemble the LH1 or LH2 chain, and store at 4℃ for later use.

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