Non-enzyme fluorescence immunoadsorption method based on DNA three-way junction auxiliary signal amplification

Abstract

The invention discloses an enzyme-free fluorescence immunoadsorption method based on DNA three-way junction auxiliary signal amplification, and belongs to the technical field of molecular biological detection. According to the invention, gold nanoparticles are taken as a carrier, an AuNPs-DNA-antibody detection probe is synthesized, an antibody is used for capturing a target object, DNA is used for amplifying a signal, the amplified signal is formed by utilizing a DNA three-way junction structure, and the signal is output through combination of generation of a G-quadruplex and a fluorescent dye. The detection on the staphylococcus aureus enterotoxin B is realized. According to the invention, high-flux, high-specificity and high-sensitivity detection of SEB can be realized. The detection limit is 7.21 ng / L, and the linear range is 0.01-10 ng / mL. The cross reaction rate to staphylococcus aureus enterotoxin A is 34.78%, and the cross reaction rates to bovine serum albumin, hemoglobin, aflatoxin B1, chloramphenicol, oxytetracycline and the like are all lower than 10%.

Description

Technical Field

[0001] The invention relates to an enzyme-free fluorescent immunoadsorption method based on DNA three-way junction-assisted signal amplification, and belongs to the technical field of molecular biology detection. Background Art

[0002] Staphylococcal enterotoxin is an exotoxin secreted by Staphylococcus aureus that can cause infection and food poisoning. More than a quarter of foodborne diseases are caused by it. Staphylococcal enterotoxin B (SEB) is a more common serotype of Staphylococcal enterotoxin, and it has good resistance to high temperature, protease, and extreme pH. Even if Staphylococci are inactivated during food processing, undenatured SEB can still cause disease, and foods with high starch, protein, and moisture content are susceptible to contamination. Therefore, the development of a method that can quickly and sensitively detect the presence of SEB in food is of great significance for preventing Staphylococcal food poisoning and controlling safety risks in food processing.

[0003] Enzyme-linked immunosorbent assay (ELISA) based on antibody-antigen recognition is a standard method for the detection of staphylococcal enterotoxin (GB 4789.10-2016). This method does not require complex instruments, cumbersome sample preparation procedures and well-trained personnel; it also has the advantages of portability, simplicity, high throughput and high specificity. However, the enzymes and markers used in the traditional ELISA method still have limitations such as high cost, easy inactivation, susceptibility to interference, and weak signal intensity, which in turn affect the repeatability and sensitivity of the method. Therefore, it is necessary to develop a new type of immunoassay method to improve the sensitivity and repeatability of traditional methods and reduce costs. This method establishes an enzyme-free, label-free fluorescent immunosorbent method in order to achieve high sensitivity, high specificity and low-cost detection of SEB content in food. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the present invention provides an enzyme-free fluorescent immunoassay method based on DNA three-way junction-assisted signal amplification, which aims to solve the limitations of the enzymes and markers used in the traditional ELISA method, such as high cost, easy inactivation, susceptibility to interference, and weak signal intensity. That is, spherical nucleic acids are used to combine the immune competition reaction with the nucleic acid isothermal amplification technology, a DNA three-way junction structure is designed for signal amplification, and the generation of G-quadruplex structure is used to interact with fluorescent dye molecules to output fluorescent signals, thereby establishing an enzyme-free and label-free fluorescent immunoassay method, which can achieve high-sensitivity, high-specificity and low-cost detection of SEB content in food.

[0005] The first technical solution provided by the present invention is a DNA composition, which comprises hairpin DNA molecules HP1, HP 2 and HP 3, wherein the nucleotide sequence of the hairpin DNA molecule HP 1 is shown in SEQ ID NO: 1, the nucleotide sequence of the hairpin DNA molecule HP 2 is shown in SEQ ID NO: 2, and the nucleotide sequence of the hairpin DNA molecule HP 3 is shown in SEQ ID NO: 3.

[0006] SEQ ID NO: 1:

[0007] 5'-TGGGGAGGGTGGGGAAAGTATACCCTCAAGCGCGCCCTCATGAGGGCAAGACTGAGGGCGCGCTTGAGGG-3';

[0008] SEQ ID NO:2:

[0009] 5'-TGGGGAGGGTGGGGAAAGCGCGCCCTCAGTCTTGCCCTCATGAGGGTATACTTGAGGGCAAGACTGAGGG-3';

[0010] SEQ ID NO:3:

[0011] 5'-TGGGGAGGGTGGGGAAGTCTTGCCCTCAAGTATACCCTCATGAGGGCGCGCTTGAGGGTATACTTGAGGG-3'.

[0012] The second technical solution provided by the present invention is a Staphylococcus enterotoxin B detection probe, wherein the probe comprises a thiol-modified Trigger DNA and a Staphylococcus enterotoxin B antibody, wherein the Trigger DNA and the Staphylococcus enterotoxin B antibody are connected to gold nanoparticles; the nucleotide sequence of the Trigger DNA is:

[0013] 5'-TGAGGGCGCGCTTGAGGGTATAC(T) 10 -SH-3'.

[0014] The third technical solution provided by the present invention is a method for preparing a SEB detection probe in certain embodiments, which specifically comprises the following steps:

[0015] (1) Synthesis of gold nanoparticles AuNPs: Heat the HAuCl4 solution to boiling, add trisodium citrate, keep boiling until an orange-red solution system is obtained, and cool to room temperature to obtain a gold nanoparticle dispersion;

[0016] (2) adjusting the pH of the gold nanoparticle dispersion, adding SEB antibody for incubation, and obtaining an AuNPs-antibody dispersion;

[0017] (3) adding the activated thiol-modified Trigger DNA to the AuNPs-antibody dispersion, mixing and freezing, dissolving the solution, adding PEG20000 and BSA for blocking, and obtaining an AuNPs-DNA-antibody dispersion;

[0018] (4) Separating the AuNPs-DNA-antibody dispersion into solid and liquid forms to obtain the probe.

[0019] In certain embodiments, in step (1), HAuCl4 is dispersed in water to prepare a 0.01% v / v HAuCl4 solution.

[0020] In certain embodiments, in step (1), trisodium citrate is added as a 1 wt % aqueous solution.

[0021] In certain embodiments, in step (1), the volume ratio of 1 wt % trisodium citrate solution to 0.01% v / v HAuCl 4 solution is 2.5 mL:100 mL.

[0022] In certain embodiments, in step (1), the mixing reaction time is 15-20 min.

[0023] In certain embodiments, in the above step (2), the pH can be specifically adjusted to 8.5-9.0.

[0024] In certain embodiments, in step (2), the incubation time is 1 hour.

[0025] In certain embodiments, in step (3), activation refers to activation of thiol-modified DNA using TCEP, wherein the molar ratio of TCEP to thiol-modified DNA is 100:1.

[0026] In certain embodiments, 15 μg of SEB antibody is added to each 1 mL of gold nanoparticle dispersion to prepare an AuNP-antibody dispersion, and then 30 nmol of Trigger DNA is added.

[0027] In certain embodiments, in step (3), the freezing reaction comprises: mixing the activated Trigger DNA and AuNPs-antibody and letting the mixture stand at -20°C for 1 hour.

[0028] In certain embodiments, in step (3), PEG 20000 is prepared as a 5 wt % aqueous solution and added until the final concentration of PEG 20000 in the solution is 0.5 wt %.

[0029] In certain embodiments, in step (3), BSA is prepared as a 10 wt % aqueous solution and added until the final concentration of BSA in the solution is 1 wt %.

[0030] In certain embodiments, in step (4), solid-liquid separation is performed by centrifugation at 13,000 rpm and 4° C. for 20 min.

[0031] In certain embodiments, the probe obtained in step (4) is dispersed in 200 μL 0.01 M PBS (containing 0.5 wt % PEG20000 and 1 wt % BSA, pH=7.4), stored at 4° C. in the dark, and used within one week.

[0032] The third technology provided by the present invention is a kit for detecting SEB, wherein the kit contains the DNA composition described in the first technical solution and the probe described in the second technical solution.

[0033] In certain embodiments, the kit further comprises a black microplate coated with SEB antigen, a SEB standard, and a fluorescent dye N-methylporphyrin dipropionate IX (NMM).

[0034] The fourth technical solution provided by the present invention is a method for detecting SEB content, which comprises the following processes: a black microplate is coated with SEB antigen and blocked; a sample to be tested and the SEB detection probe described in the second technical solution are added to the black microplate, and after incubation, the sample is washed and patted dry; the DNA composition described in the first technical solution is added to react; fluorescent dye NMM is added to react and fluorescence detection is performed; the difference between the fluorescence value obtained by the detection and the fluorescence value when the SEB content is 0 is brought into the standard curve to calculate the SEB concentration.

[0035] In certain embodiments, the standard curve is y(au)=2593.81x(ng / mL)+6367.95, wherein y(au) is the difference between the fluorescence value detected and the fluorescence value when the SEB content is 0, and x(ng / mL) is the logarithm of the SEB content.

[0036] In certain embodiments, the black microplate is coated with antigen by adding 2 μg / mL SEB, coated overnight at 4° C., and blocked with 2 wt % BSA for 1 h.

[0037] In certain embodiments, the SEB detection probe carrying the Trigger DNA is diluted with a buffer solution and then added to a black microplate; the buffer solution comprises 0.01 M PBS, 0.5 wt % PEG20000, and 1 wt % BSA; and the dilution is 40 times.

[0038] In certain embodiments, the hairpin DNA molecules HP 1, HP 2, and HP 3 are added at a concentration of 500 nmol / L, and the reaction is carried out at 37° C. for 1.5 h.

[0039] In certain embodiments, the fluorescent dye NMM is added at a concentration of 8 μmol / L, and the reaction is carried out at 37° C. for 15 min.

[0040] In certain embodiments, the fluorescence intensity signal is detected using an excitation wavelength of 399 nm / emission wavelength of 610 nm.

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

[0042] The present invention uses gold nanoparticles as carriers to synthesize AuNPs-DNA-antibody detection probes, wherein the antibody is used to capture the target, and the DNA is used to amplify the signal, and the signal is amplified by forming a DNA three-way junction structure, and the signal is output by combining with a fluorescent dye through the generation of a G-quadruplex, thereby realizing the detection of Staphylococcus aureus enterotoxin B.

[0043] The present invention has good detection specificity, stability and accuracy for SEB. High-throughput, high-specificity and high-sensitivity detection of SEB can be achieved without complex equipment and skilled operators. The detection limit is 7.21 ng / L, and the linear range is 0.01-10 ng / mL. The cross-reaction rate to Staphylococcus aureus enterotoxin A is 34.78%, and the cross-reaction rates to bovine serum albumin, hemoglobin, aflatoxin B1, chloramphenicol, oxytetracycline, etc. are all less than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the principle of the present invention.

[0045] Figure 2 UV images of AuNPs and AuNPs-DNA-antibody.

[0046] Figure 3 To verify the feasibility of the scheme, (A) fluorescence signal verification; (B) agarose gel electrophoresis verification

[0047] Figure 4 Condition optimization: (A) coating antigen and probe concentration optimization; (B) reaction time; (C) hairpin DNA concentration optimization; (D) fluorescent dye concentration optimization;

[0048] Figure 5 To detect the standard curve, SEB concentrations were set to 0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, and 10 ng / mL.

[0049] Figure 6For validation of the selectivity of the scheme, Staphylococcus aureus enterotoxin A, aflatoxin B1, bovine serum albumin, bovine hemoglobin, chloramphenicol, and oxytetracycline were used as references. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following is a detailed description of the present invention in conjunction with the following specific embodiments. Figures 1 to 6 , the present invention is further described in detail.

[0051] Materials used in the examples:

[0052] Reagents and Materials: Staphylococcus aureus enterotoxin B (SEB) was purchased from Beijing Bio-Innovation Technology Co., Ltd.; Staphylococcus aureus enterotoxin B antibody was purchased from Beijing Bio-Science Biotechnology Co., Ltd.; trisodium citrate, Tween 20, bovine serum albumin (BSA), and polyethylene glycol 20000 (PEG 20000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0053] Coating buffer (0.05M CBS, pH 9.6), PBST (0.01M PBS, 0.05% Tween 20), 0.01M PBS (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, and 8mM K2HPO4, pH 7.2), reaction buffer (10mMTris-HCl, 50mM KCl, 5mM MgCl2, pH=8.0). All oligonucleotides were synthesized, modified and purified by Sangon (Shanghai, China).

[0054] Instruments: H1M1 enzyme-linked immunosorbent analyzer (Guangzhou Darui Biotechnology Co., Ltd.); T9-UV-vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd.); shaking incubator (Thermo Fisher Scientific, USA).

[0055] Example 1 Synthesis of detection probe

[0056] (1) Preparation of AuNPs:

[0057] AuNPs were prepared by citrate reduction method. 100 mL of 0.01% HAuCl4 was heated to boiling on a magnetic stirrer. 2.5 mL of freshly prepared 1% trisodium citrate was then added to the boiling solution under vigorous stirring. The mixture was boiled continuously for about 15 min until the color turned orange-red. The solution was then cooled to ambient temperature under slow stirring, added to 100 mL of dispersion, and stored at 4 °C in the dark.

[0058] (2) Synthesis of AuNPs-DNA-antibody detection probe:

[0059] 1) 1 mL of AuNPs dispersion (pH 8.5-9.0) was incubated with 15 μg of SEB antibody at room temperature for 1 h with gentle stirring to obtain a mixed solution.

[0060] 2) Use 50 μL of TCEP to activate the thiol-modified Trigger DNA (sequence: 5'-TGAGGGCGCGCTTGAGGGTATAC(T) 10 -SH-3')

[0061] 3) Add the Trigger DNA from step 2) to the mixed solution in step 1) and let stand at -20°C for 1h. After melting, add 5wt% PEG 20000 (final concentration is 0.5wt%) to the mixed solution. Finally, add 10% w / v BSA to block the remaining active sites on the surface. In order to remove excess antibodies and oligonucleotides, the solution was centrifuged at 13000rpm and 4°C for 20 minutes to obtain the AuNPs-DNA-antibody detection probe. The probe was then resuspended in 200μL storage buffer (0.01M PBS, 0.5wt% PEG 20000, 1wt% BSA) and stored at 4°C in the dark.

[0062] The results of UV-vis spectroscopy were as follows: Figure 2 As shown in the figure, the maximum UV absorption peak of AuNPs is at 520nm, while the maximum UV absorption peak of Ab-AuNPs-DNA probe is red-shifted to 528nm, and characteristic absorption peaks of proteins and nucleic acids appear, indicating that the antibody and Trigger DNA have been successfully modified on the surface of AuNPs.

[0063] Example 2: Feasibility Verification

[0064] (1) Gel electrophoresis verification

[0065] First, prepare 3.5% agarose gel by weighing 2.8 g agarose powder and dissolving it in 80 mL 0.5×TBE buffer and heating it in a microwave for 2 min until it is completely dissolved. Pour the solution into a gel-making plate and insert a comb, and let it stand until it solidifies.

[0066] The prepared sample solution and the loading buffer containing the staining solution were mixed thoroughly at a volume ratio of 5:1, and then added into the wells in the order (1: HP 1; 2: HP 1+HP 2+HP 3; 3: Trigger DNA+HP 1+HP 2+HP 3; M: marker); the nucleotide sequence of the Trigger DNA was: 5'-TGAGGGCGCGCTTGAGGGTATAC(T) 10 -3'; The nucleotide sequence of the hairpin DNA molecule HP1 is:

[0067] 5'-TGGGGAGGGTGGGGAAAGTATACCCTCAAGCGCGCCCTCATGAGGGCAAGACTGAGGGCGCGCTTGAGGG-3';

[0068] The nucleotide sequence of the hairpin DNA molecule HP 2 is:

[0069] 5'-TGGGGAGGGTGGGGAAAGCGCGCCCTCAGTCTTGCCCTCATGAGGGTATACTTGAGGGCAAGACTGAGGG-3';

[0070] The nucleotide sequence of the hairpin DNA molecule HP 3 is:

[0071] 5'-TGGGGAGGGTGGGGAAGTCTTGCCCTCAAGTATACCCTCATGAGGGCGCGCTTGAGGGTATACTTGAGGG-3'.

[0072] The incubation conditions were 37°C for 1 hour. The electrophoresis conditions were 0.5×TBE electrophoresis buffer, 120V, 80mA, 1 hour. After completion, the gel imaging system was used for analysis.

[0073] The results are as follows Figure 3 As shown in A. Lane 2 shows the case where there is no Trigger DNA in the reaction system, and it can be seen that the hairpin DNAs failed to open each other. Trigger DNA was added to lane 3, and a high molecular weight DNA band was generated, indicating that the hairpin DNAs were successfully opened and hybridized with each other to form a high molecular weight DNA product. The successful progress of the reaction was verified.

[0074] (2) Fluorescence spectroscopy analysis

[0075] 0-1.5 μM TriggerDNA was added to the system containing equal amounts of hairpin DNA molecules HP 1, HP 2, and HP 3, respectively, and incubated at 37°C for 1 hour. To ensure the formation of the hairpin structure, the hairpin DNA molecules HP 1, HP 2, and HP 3 were heated to 95°C for 10 minutes before use, and then cooled to room temperature. The fluorescence spectrum was scanned from 550nm to 750nm using a fluorescence spectrophotometer, with an excitation wavelength of 399nm.

[0076] The results are as follows Figure 3 As shown in B, the fluorescence intensity was significantly improved after adding 0.5 μM Trigger DNA. As the concentration of Trigger DNA increased, the fluorescence intensity also increased. The results were consistent with the detection principle.

[0077] Example 3: Optimization of detection conditions

[0078] The detection steps are as follows:

[0079] 100 μL of SEB was added to a 96-well black microplate and coated overnight at 4°C. After washing three times with 0.05% PBST, 200 μL of 2% BSA was added and blocked at 37°C for 1 hour. Then, 50 μL of 10 ng / mL SEB standard solution and 50 μL of the diluted AuNP-DNA-antibody detection probe prepared in Example 1 were added respectively, and incubated at 37°C for 1 hour for immune competition reaction. After washing, 100 μL of a certain concentration of hairpin DNA molecules HP 1, HP 2, and HP 3 were added and reacted at 37°C. Finally, 100 μL of N-methylporphyrin dipropionic acid IX (NMM) was added and reacted at 37°C for 15 minutes. The fluorescence intensity was measured at an excitation wavelength of 399 nm / emission wavelength of 610 nm.

[0080] (1) Optimization of coating antigen and probe concentration

[0081] According to the above detection steps, the concentrations of coated antigen and probe were optimized by chessboard method. The concentrations of coated antigen were set to 0.5μg / mL, 1μg / mL, 2μg / mL and 2.5μg / mL, and the dilution ratios of detection probe were 1:20, 1:40 and 1:80, respectively. The enhancement rate (F0-F) / F0 (F0: fluorescence intensity when SEB concentration is 0ng / mL, F: fluorescence intensity when SEB concentration is 10ng / mL) was used as the evaluation index of the optimization results.

[0082] The results are as follows Figure 4 As shown in A, the optimal concentration of the coating antigen was set to 2 μg / mL, and the optimal dilution of the detection probe was set to 1:40.

[0083] (2) Optimization of the concentrations of hairpin DNA molecules HP 1, HP 2, and HP 3

[0084] According to the above detection steps, the concentrations of the hairpin DNA molecules HP 1, HP 2, and HP 3 were changed to 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, and 2 μM, respectively, and the other conditions remained unchanged for detection.

[0085] The inhibition rate was used as the evaluation standard. Figure 4 As shown in B, 0.5 μM was selected as the optimal condition, at which time the inhibition rate reached the maximum.

[0086] (3) Optimization of signal amplification reaction time

[0087] According to the above detection steps, the signal amplification reaction time was changed to 60min, 90min, 120min and 150min respectively, and the other conditions remained unchanged for detection.

[0088] The inhibition rate was used as the evaluation index. Figure 4 As shown in C, 90 min was selected as the optimal reaction time.

[0089] (4) Dye concentration optimization

[0090] According to the above detection steps, the concentration of dye NMM was changed to 2μM, 5μM, 8μM, and 10μM respectively, and the other conditions remained unchanged for detection.

[0091] The inhibition rate was used as the evaluation index. Figure 4 As shown in D, 8 μM was selected as the optimal dye concentration.

[0092] Example 4: Linear range verification

[0093] Based on Example 3, the optimal data obtained by optimization was used to set the concentration of coated antigen SEB to 2 μg / mL, the dilution multiple of AuNP-DNA-antibody detection probe to 40 times, the concentration of hairpin DNA molecules to 0.5 μM, the signal amplification reaction time to 90 min, and the amount of dye NMM to 8 μM. Under these conditions, a series of diluted SEB standard sample concentrations (0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5 ng / mL) were detected. Fluorescence was obtained at 399 nm / 610 nm.

[0094] like Figure 5 As shown, a standard curve with a linear range of 0.01-10 ng / mL was obtained, and the linear equation was y=2593.81x+6367.95, R 2 =0.96, and the limit of detection (LOD) was 7.21 ng / L.

[0095] Example 5: Specificity

[0096] Staphylococcal enterotoxin A, bovine serum albumin, hemoglobin, aflatoxin B1, chloramphenicol, and oxytetracycline, common pollutants in food and structural analogs of SEB, were selected as test samples to evaluate the specificity of the detection method of the present invention.

[0097] Based on Example 3, the best data obtained by optimization was used and no steps were omitted for testing. Figure 6 As shown, except for SEA, which has a very similar structure, other common pollutants or structural analogs show low cross-reactivity rates.

[0098] Example 6 Accuracy

[0099] Two common pre-prepared foods on the market, pickled fish and lion's head, were selected as actual samples, and different concentrations of SEB standard (0.05, 0.5, 5 ng / mL) were added as test samples to evaluate the accuracy. The detection method and conditions refer to the optimal conditions of Example 3.

[0100] As shown in Table 1, the recovery rate obtained ranged from 87.35% to 118.55%, and the RSD was substantially less than 10%. The results show that the method for detecting SEB provided by the present invention has good accuracy.

[0101] Table 1: Recovery verification results

[0102]

[0103] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Staphylococcal enterotoxin B detection probe, characterized in that: The probe comprises a thiol-modified Trigger DNA and a Staphylococcus enterotoxin B antibody, wherein the Trigger DNA and the Staphylococcus enterotoxin B antibody are connected to gold nanoparticles; the nucleotide sequence of the thiol-modified Trigger DNA is: 5'-TGAGGGCGCGCTTGAGGGTATAC(T) 10 -SH-3'.

2. A method for preparing a SEB detection probe, characterized in that: The specific steps include: (1) Synthesis of gold nanoparticles AuNPs: Heat the HAuCl4 solution to boiling, add trisodium citrate solution, keep boiling until an orange-red solution system is obtained, and cool to room temperature to obtain a gold nanoparticle dispersion; (2) adjusting the pH of the gold nanoparticle dispersion, adding SEB antibody for incubation, and obtaining an AuNPs-antibody dispersion; (3) adding the activated thiol-modified Trigger DNA to the AuNPs-antibody dispersion, mixing and freezing, dissolving the solution, adding PEG20000 and BSA for blocking, and obtaining an AuNPs-DNA-antibody dispersion; (4) performing solid-liquid separation on the obtained AuNPs-DNA-antibody dispersion to obtain a probe; The nucleotide sequence of the thiol-modified Trigger DNA is: 5'-TGAGGGCGCGCTTGAGGGTATAC(T) 10 -SH-3'.

3. The method according to claim 2, characterized in that 15Nμg SEB antibody was added to N mL gold nanoparticle dispersion to prepare AuNP-antibody dispersion, and then 30N nmol Trigger DNA was added.

4. The method according to claim 2, characterized in that: The activation refers to activating the thiol-modified Trigger DNA using TCEP, wherein the molar ratio of TCEP to the thiol-modified Trigger DNA is 100:

1.

5. The method according to claim 2, characterized in that: The freezing reaction includes: mixing the activated Trigger DNA and AuNPs-antibody, and then standing at -30 to 15° C. for 55 to 70 hours.

6. The method according to claim 2, characterized in that In step (3), in the AuNPs-DNA-antibody dispersion, the final concentration of PEG 20000 is 0.5wt%; and the final concentration of BSA is 1wt%.

7. A kit for detecting SEB, characterized in that: The kit contains a DNA composition, the probe according to claim 1, a black microplate coated with SEB antigen, a SEB standard and a fluorescent dye N-methylporphyrin dipropionate IX, wherein the DNA composition includes hairpin DNA molecules HP 1, HP 2 and HP 3, the nucleotide sequence of the hairpin DNA molecule HP 1 is shown in SEQ ID NO: 1, the nucleotide sequence of the hairpin DNA molecule HP 2 is shown in SEQ ID NO: 2, and the nucleotide sequence of the hairpin DNA molecule HP 3 is shown in SEQ ID NO:

3.

8. A method for detecting SEB content, characterized in that: The method comprises the following steps: a black microplate is coated with SEB antigen and sealed; a sample to be tested and the SEB detection probe according to claim 1 are added to the black microplate, and after incubation, the plate is washed and patted dry; A DNA composition is added to react; a fluorescent dye N-methylporphyrin dipropionic acid IX is added to react and then fluorescence detection is performed; the difference between the fluorescence value obtained by the detection and the fluorescence value when the SEB content is 0 is brought into the standard curve to calculate the SEB concentration.

9. The method according to claim 8, characterized in that The concentration of SEB coated antigen in the detection sample solution is 0.5-2.5 μg / mL, the concentration of hairpin DNA molecules HP 1, HP 2, and HP 3 in the detection sample solution is 250-2000 nmol / L, the concentration of the fluorescent dye N-methylporphyrin dipropionic acid IX in the detection sample solution is 2-10 μmol / L, and the probe dilution multiple is 40 times.

10. A DNA composition, characterized in that The composition comprises hairpin DNA molecules HP 1, HP 2 and HP 3, wherein the nucleotide sequence of the hairpin DNA molecule HP 1 is shown in SEQ ID NO: 1, the nucleotide sequence of the hairpin DNA molecule HP 2 is shown in SEQ ID NO: 2, and the nucleotide sequence of the hairpin DNA molecule HP 3 is shown in SEQ ID NO: 3.

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