Multi-target fluorescence immunoassay method based on TMSD-DNAzyme

Abstract

The invention discloses a multi-target fluorescence immunoassay method based on TMSD-DNAzyme, which is used for simultaneously detecting staphylococcus aureus enterotoxin B (SEB) and GII type norovirus (NoV), and comprises the following steps: S1, coating a 96-hole black microplate with an SEB capture antibody, and sealing; s2, a diluted immune probe and a sample to be detected are added, a sandwich structure is formed through an immune reaction, and the immune probe is formed by coupling an SEB detection antibody and a Trigger chain on the surface of a gold nanoparticle; according to the invention, by designing two sets of independent signal amplification channels which can run in parallel, the detection of SEB protein and NoV nucleic acid is completed in one reaction hole at the same time, compared with a traditional mode needing two independent ELISA detection, the method has the advantages that the detection process is simplified by nearly one time, the real'one-time detection and double results' is realized, and the detection efficiency is greatly improved. The detection flux is greatly improved, and about 50% of sample, reagent and time cost is saved.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and detection technology, and particularly relates to a multi-target fluorescence immunoassay method based on TMSD-DNAzyme. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) has become the gold standard for detecting protein toxoids (such as SEB) due to its high specificity, ease of operation, and ability to achieve high-throughput detection. It has also been incorporated into national standards in many countries (such as GB4789.10-2016). Traditional ELISA technology relies on the reaction between enzyme-labeled antibodies and substrates to generate a signal, but its sensitivity is limited by the enzyme's catalytic efficiency, and the enzyme is prone to inactivation and has a high cost.

[0003] In the detection of viruses such as NoV, nucleic acid-based detection methods are the mainstream technology due to the difficulty in in vitro culture. Although the development of technologies such as loop-mediated isothermal amplification (LAMP) has made rapid on-site detection possible, these nucleic acid detection technologies are usually independent of immunoassay techniques for protein toxins, resulting in two separate and time-consuming pretreatment and detection procedures for different types of hazards (bacterial toxins and viruses) in the same sample.

[0004] Current ELISA technology typically detects only a single target at a time. When dealing with the need to simultaneously detect multiple targets (such as SEB and norovirus) in complex samples, multiple operations are required, leading to high sample consumption, long detection times, high costs, and susceptibility to cross-reactions and human error. Repeated detection is not only inefficient but may also affect the accuracy and reliability of results due to antibody cross-reactions or signal interference. Furthermore, current technologies struggle to balance high sensitivity and ease of operation in multi-target detection, limiting their widespread application in clinical diagnostics, food safety, and environmental monitoring. Summary of the Invention

[0005] To overcome the aforementioned deficiencies of the prior art, embodiments of the present invention provide a multi-target fluorescence immunoassay method based on TMSD-DNAzyme, which solves the problems of existing ELISA technology, which can usually only detect a single target at a time. When faced with the need to detect multiple targets (such as SEB and norovirus) in complex samples at the same time, multiple operations are required, resulting in large sample consumption, long detection time, high cost, and easy introduction of cross-reaction and human error.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A multi-target fluorescence immunoassay method based on TMSD-DNAzyme for the simultaneous detection of Staphylococcus aureus enterotoxin B (SEB) and GII norovirus (NoV) includes the following steps:

[0008] S1. Coat the SEB capture antibody onto a 96-well black microplate and block it;

[0009] S2. Add the diluted immune probe and the sample to be tested, and form a sandwich structure through an immune reaction. The immune probe is a gold nanoparticle surface coupled with SEB detection antibody and Trigger chain (sequence: SH-TTT TTT TTT TTT TTT ATGAAG ATG GAG CTG GAG TG).

[0010] S3. Add the nucleic acid sample to be tested and the functional nucleic acid mixture, and incubate at 37°C in the dark to achieve TMSD-activated DNAzyme cleavage and CHA cascade amplification. The functional nucleic acid mixture contains P1-P3 recognition sequences, HP-A / HP-B DNAzyme substrates and HP1-HP6CHA hairpin structures.

[0011] S4. Fluorescence intensity was detected at 490nm / 520nm (FAM channel) and 532nm / 568nm (Cy3 channel) using an enzyme-linked immunosorbent assay (ELISA) reader to achieve quantitative analysis of SEB and NoV.

[0012] Preferably, the P1 sequence is 5'-TCA CTC GAC GCG GTC GAA TAG TGT GTC GCT C-3', the P2 sequence is 5'-AAG TAC GTC TCC GAG CCC ATC TTC AT-3', and the P3 sequence is 5'-CAC TCC AGC TCG GTCGAA CGT TGAACC CAT A-3'.

[0013] Preferably, the HP-A sequence is 5'-CTG CAT CCT TTT TTG AGC GAC ACA CTA T / rA / GGAC GTA CTT TTT TGG ATG CAG-3', and the HP-B sequence is 5'-ATG GGT CTC ACT ATG GGT TCAACG T / rA / G GAC GTA CTT-3'.

[0014] Preferably, the HP1 sequence is 5'-FAM-TAT AGT GTG TCG CTC AAAAAA GGA TGC TCCAAT CAC AAC TGC ATC CTT TTT TGA GCG ACA-BHQ1-3', and the HP4 sequence is 5'-Cy3-CGT TGAACCCAT AGT GAG ACC CAT ATG GGT CAA GAC ATG GGT CTC ACT-BHQ2-3'.

[0015] Preferably, in the functional nucleic acid mixture, the concentrations of P1 and P2 are both 1000 nmol / L, the concentration of P3 is 800 nmol / L, the concentration of HP-A is 1200 nmol / L, the concentration of HP-B is 1000 nmol / L, and the concentrations of HP1-HP6 are all 500 nmol / L.

[0016] Preferably, the preparation of the immune probe includes the steps of gold nanoparticle synthesis, co-conjugation of SEB detection antibody with a final concentration of 18 μg / mL with the Trigger chain, PEG20000 and BSA blocking, and centrifugation purification.

[0017] Preferably, the immune reaction conditions are incubation at 37°C for 1 hour, and the signal amplification reaction conditions are incubation at 37°C in the dark for 2 hours.

[0018] Preferably, the coating buffer is a 0.05M carbonate buffer at pH 9.6, and the reaction buffer is a pH 8.0 buffer containing 10 mmol / L Tris-HCl, 50 mmol / L KCl, and 5 mmol / L MgCl2.

[0019] Preferably, the detection linear range of SEB is 0.01–10 ng / mL, and the detection linear range of NoV nucleic acid is 5–250 nmol / L.

[0020] Preferably, the immune probes are diluted with 0.01MPBS at a volume ratio of 1:20 before use, and HP-A to HP6 are heated at 95°C for 10 minutes and then naturally cooled to room temperature before use.

[0021] The technical effects and advantages of the multi-target fluorescent immunoassay method based on TMSD-DNAzyme of this invention are as follows:

[0022] 1. This invention designs two independent but parallel signal amplification pathways (triggered by the Trigger and Target chains respectively) to simultaneously detect SEB protein and NoV nucleic acid in a single reaction well. Experiments have shown that after a signal amplification reaction lasting approximately 2 hours, fluorescence signals from two channels, 520 nm (FAM, corresponding to SEB) and 568 nm (Cy3, corresponding to NoV), can be read in the same microwell.

[0023] Compared to the traditional method that requires two independent ELISA tests, this method simplifies the testing process by nearly half, achieving true "one test, two results," greatly increasing throughput and saving approximately 50% of sample, reagent, and time costs.

[0024] 2. This invention employs a cascade amplification strategy consisting of DNAzyme cleavage reaction and catalytic hairpin self-assembly, providing efficient and specific dual signal amplification for each target analyte. Experimental data show that the method achieves a detection limit (LOD) of 1.7 pg / mL for SEB and 2.42 nmol / L for NoV nucleic acid.

[0025] The sensitivity of this method for SEB (1.7 pg / mL) is significantly better than the national standard ELISA method (LOD 250 pg / mL), and also better than the single-target method based on CHA constructed in Chapter 2 of this paper (LOD 7.21 pg / mL). This indicates that while successfully integrating multi-target detection capabilities, this invention not only does not sacrifice sensitivity, but also maintains a high level of sensitivity through a more efficient cascade amplification design.

[0026] 3. This invention achieves highly specific recognition of the Trigger and Target chains through three carefully designed recognition sequences (P1, P2, P3). Selectivity experiments show that for SEB detection, except for a 38% cross-reactivity with structurally similar Staphylococcus aureus enterotoxin A (SEA), there is no significant cross-reactivity with common food contaminants and structural analogs such as aflatoxin B1, chloramphenicol, and oxytetracycline. For NoV nucleic acid detection, the relative fluorescence intensity of the designed single-base mismatch (SM), double-base mismatch (DM), triple-base mismatch (TM), and complete mismatch (CM) sequences is significantly lower than that of the completely matched target sequences, demonstrating the accuracy of the nucleic acid recognition process.

[0027] This specificity ensures that, in complex sample matrices, the two signal channels of SEB and NoV can respond independently and accurately to their respective targets, effectively avoiding misjudgments caused by cross-reactions and meeting the reliability requirements of actual sample detection. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention;

[0029] Figure 2 This is a feasibility analysis diagram of the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention;

[0030] Figure 3 This invention optimizes the key parameters of the detection system for the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention. Figure 1 ;

[0031] Figure 4This invention optimizes the key parameters of the detection system for the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention. Figure 2 ;

[0032] Figure 5 This is an optimized reaction time diagram of the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention;

[0033] Figure 6 This is a standard curve of SEB and NoV detection for the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention;

[0034] Figure 7 This figure shows the experimental results of the stability and selectivity of the multi-target fluorescent immunoassay method based on TMSD-DNAzyme proposed in this invention.

[0035] Legend:

[0036] Figure 2 (A) Fluorescence spectral characterization diagram; (B) Agarose gel electrophoresis analysis; (C) Fluorescence spectral characterization diagram; (D) Agarose gel electrophoresis analysis;

[0037] Figure 3 (A) Optimization of coating antibody concentration and probe dilution factor; (B) Optimization of P1 and P2 concentrations; (C) Optimization of P3 concentration;

[0038] Figure 4 (A) HP-A concentration optimization; (B) HP-B concentration optimization; (C) HP1, HP2, HP3 concentration optimization; (D) HP4, HP5, HP6 concentration optimization;

[0039] Figure 5 (A) Target reaction system; (B) Trigger reaction system;

[0040] Figure 6 (A) Standard curve of SEB; (B) Standard curve of NoV;

[0041] Figure 7 (A) Stability test results; (B) SEB selectivity test; (C) Nucleic acid selectivity test. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0043] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0044] Basic experimental conditions:

[0045] Main instruments:

[0046] 96-well black microplate (Costar, USA);

[0047] Multifunctional microplate reader (BioTek Synergy H1, USA);

[0048] Constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd., HWS-24);

[0049] Micropipettes (Eppendorf, Germany);

[0050] High-speed centrifuge (Eppendorf, Germany);

[0051] Ultraviolet-visible spectrophotometry (Shimadzu, Japan, UV-1800);

[0052] Precision pH meter (Thermo Scientific, USA).

[0053] Reagents and materials:

[0054] Chloroauric acid (HAuCl4・3H2O), trisodium citrate, magnesium chloride (MgCl2), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), Tris base, Tween 20, bovine serum albumin (BSA, 96% purity), polyethylene glycol 20000 (PEG20000), and tris(2-carbonylethyl) phosphate hydrochloride (TCEP) were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0055] The oligonucleotide sequences (HPLC purity, see Table 1) were synthesized by Shanghai Sangon Biotech Co., Ltd., of which HP-A and HP-B contain RNA bases ( / rA / ).

[0056] Staphylococcus aureus enterotoxin B (SEB) standard, Staphylococcus aureus enterotoxin A (SEA) standard, SEB capture antibody, and SEB detection antibody were purchased from Beijing Bio-Innovation Technology Co., Ltd.

[0057] Aflatoxin B1 (AFB1), chloramphenicol (CAP), and oxytetracycline (OTC) standards were purchased from Manhag (Shanghai) Biotechnology Co., Ltd.

[0058] The water used in the experiment was Milli-Q ultrapure water (resistivity ≥18.2 MΩ・cm).

[0059] The oligonucleotide sequences used in this embodiment are shown in the table below:

[0060] name Sequence (5' end - 3' end) Function Description P1 TCA CTC GAC GCG GTC GAA TAG TGT GTC GCT C The target chain, together with P2, activates DNAzyme. P2 AAG TAC GTC TCC GAG CCC ATC TTC AT Identify the Target or Trigger chain and coordinate with P1 or P3 respectively. P3 CAC TCC AGC TCG GTC GAA CGT TGAACC CAT A Identify the trigger chain and activate DNAzyme together with P2. HP-A CTG CAT CCT TTT TTG AGC GAC ACA CTA T / rA / G GACGTA CTT TTT TGG ATG CAG DNAzyme substrates are cleaved by the P1 / P2-Target complex, releasing the T1 strand. HP-B ATG GGT CTC ACT ATG GGT TCAACG T / rA / G GAC GTA CTTTTT TAG ACC CAT DNAzyme substrates are cleaved by the P2 / P3-Trigger complex, releasing the T2 strand. HP1 FAM-TAT AGT GTG TCG CTC AAAAAA GGA TGC TCC AATCAC AAC TGC ATC CTT TTT TGA GCG ACA-BHQ1 CHA-reactive hairpin structure 1, containing a FAM fluorescent group and a BHQ1 quencher group. HP2 TCAAAAAAG GAT GCA GTT GTG ATT GGA GCG ACA CAC TATATC CAA TCA CAA CTG CAT CCT CHA reaction hairpin structure 2 HP3 AGT TGT GAT TGG ATA TAG TGT GTC GCG CAT CCT TTTTTG AGC GAC ACA CTA TAT CCAAT CHA reaction hairpin structure 3 HP4 Cy3-CGT TGAACC CAT AGT GAG ACC CAT ATG GGT CAAGAC ATG GGT CTC ACT-BHQ2 CHA-reactive hairpin structure 4, containing a Cy3 fluorescent group and a BHQ2 quencher group. HP5 AGT GAG ACC CAT GTC TTG ACC CAT ATG GGT TCAACGATG GGT CAA GAC CHA reaction hairpin structure 5 HP6 GTC TTG ACC CAT CGT TGAACC CAT ATG GGT CT C ACTATG GGT TCA ACG CHA reaction hairpin structure 6 Trigger SH-TTT TTT TTT TTT TTT ATG AAG ATG GAG CTG GAG TG Immunoplasmic probe DNA Target-RNA AUG AAG AUG GCG UCG AGU GA NoV target RNA Target-RNA AUG AAG AUG GCG UCG AGU GA NoV target DNA

[0061] (Note: / rA / represents an RNA base)

[0062] Table 1

[0063] Solution preparation:

[0064] 0.01M PBS (pH 7.4): Weigh out NaCl (8.0g), KCl (0.2g), Na2HPO4 (1.44g), and KH2PO4 (0.24g), dissolve them in 1L of ultrapure water, and adjust the pH to 7.4 with HCl or NaOH;

[0065] Coating buffer (0.05M CB, pH 9.6): Weigh 2.93g NaHCO3 and 1.59g Na2CO3 and dissolve them in 1L of ultrapure water;

[0066] PBST washing buffer: Measure 1L of 0.01M PBS, add 0.5mL of Tween 20, and mix well;

[0067] Reaction buffer (pH 8.0): Measure 10 mM Tris-HCl buffer, add KCl to a final concentration of 50 mM, and MgCl2 to a final concentration of 5 mM;

[0068] Blocking solution: 0.01M PBS solution containing 2% (w / v) BSA;

[0069] Probe storage solution: 0.01M PBS solution containing 0.5% PEG20000 and 1% BSA.

[0070] refer to Figures 1-2 The experimental steps in this embodiment are as follows:

[0071] Gold nanoparticles were synthesized using the citric acid reduction method: A concentration of 0.01% (w / v) Heat the solution to boiling point, then quickly add the solution while stirring vigorously. A 1% (w / v) trisodium citrate solution was prepared; after the solution changed from pale yellow to wine red, reflux and stirring were continued for 15 minutes. Heating was then stopped, and the solution was stirred and cooled to room temperature. The volume was then made up with ultrapure water to the required level. Store at 4℃ away from light. The absorbance at 520 nm was measured using UV-Vis spectroscopy. Calculate the concentration.

[0072] To determine the optimal amount of antibody for SEB detection, 1 mL of the above AuNPs was taken, and the pH was adjusted to 8.5-9.0 with 0.1 Mk2CO3. Different volumes of [unspecified substance] were then added. The SEB detection antibody was used to achieve a final concentration of... Within the specified range, after standing at room temperature for 5 minutes, add to each tube A 10% NaCl solution, after being mixed and allowed to stand in the dark for 2 hours, was observed to have a minimum antibody concentration that maintained a red color without turning blue. Add 20% to this, resulting in a final concentration of The SEB detection antibody was used as the formal experimental dosage.

[0073] Towards Add to AuNPs SEB detection antibodies (corresponding) Final concentration), incubate at room temperature with gentle shaking for 1 hour; simultaneously, Trigger dry powder Dissolve in ultrapure water and add Prepare a 100 mg / L MTCEP solution and reduce it at room temperature for 15 minutes. After the reduction is complete, add the entire Trigger solution to the Ab-AuNPs solution, mix well, and freeze at -20°C for 1 hour. After freezing, allow it to thaw naturally at room temperature.

[0074] Add PEG20000 and BSA to the thawed solution to final concentrations of 0.5% and 1%, respectively, and incubate at room temperature for 30 minutes. Then centrifuge at 4°C and 13000 rpm for 20 minutes, discard the supernatant, and use... The precipitate was resuspended in the probe storage solution, and the mixture was washed three times by centrifugation. Finally, the purified Ab-AuNPs-DNA immunoprobe was resuspended in... Store the probe in storage solution at 4°C, protected from light.

[0075] Further, the SEB capture antibody was diluted with coating buffer (0.05 MCB, pH 9.6) to... Add to each well of the 96-well black microplate After dilution, the capture antibody solution was sealed with sealing film and incubated overnight at 4°C (12-16 hours). After incubation, the coating solution in the wells was discarded, and the antibody was added to each well. Add PBST washing buffer, let stand for 1 minute, discard, and pat dry on absorbent paper. Repeat washing 3 times; then add PBST washing buffer to each well. Incubate the microplate with 2% BSA blocking solution at 37°C for 1 hour. After discarding the blocking solution, wash the microplate three times with PBST as described above. The washed microplate can be used immediately or sealed and stored at 4°C (it is recommended to use it within one week).

[0076] Furthermore, the prepared Ab-AuNPs-DNA immunoprobe was diluted with 0.01M PBS at a volume ratio of 1:20, and then added sequentially to each well of the coated and blocked microplate. Diluted immune probes and SEB standard solution (prepared with 0.01M PBS at concentration gradients of 0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, 10 ng / mL) or the food sample to be tested, sealed with sealing film, incubated at 37°C for 1 hour; after incubation, discard the liquid in the wells, and add [the appropriate amount of solution] to each well. Use PBST washing solution, let stand for 1 minute, discard and pat dry. Repeat washing 5 times to completely remove unbound immune probes.

[0077] Furthermore, according to the following final concentration, the concentration is... Tris-HCl, concentration of KCl, concentration of In a reaction buffer solution with pH 8.0, MgCl2 is used to premix the following functional nucleic acid components: P1 (1000 nmol / L), P2 (1000 nmol / L), P3 (800 nmol / L), HP-A (1200 nmol / L), HP-B (1000 nmol / L), and HP1 to HP6, each at 500 nmol / L. HP-A to HP6 need to be heated in a 95°C water bath for 10 minutes before use, and then allowed to cool slowly to room temperature (about 2 hours) after the water bath is turned off to ensure the formation of the correct hairpin structure.

[0078] Add sequentially to each well of the microplate after the immune reaction washing. NoV target RNA solution (prepared with RNase-free water at concentration gradients of 0, 5, 10, 25, 50, 75, 100, and 250 nmol / L) or the nucleic acid sample to be tested, and The prepared functional nucleic acid mixture was sealed with a sealing film and incubated at 37°C in the dark for 2 hours to activate the DNAzyme cleavage and catalytic hairpin self-assembly (CHA) cascade amplification reaction.

[0079] Furthermore, after the signal amplification reaction is complete, no additional processing is required; the fluorescence intensity can be directly detected using a multi-functional microplate reader: the SEB detection channel is set to excitation wavelength 490nm and emission wavelength 520nm, and the fluorescence intensity of FAM on HP1 is read. The NoV detection channel was set to excitation wavelength 532nm and emission wavelength 568nm to read the fluorescence intensity of Cy3 on HP4. ).

[0080] The fluorescence intensity measured in blank wells without SEB and NoV targets was used as the background value. , ), calculate net fluorescence intensity: , ; respectively using the logarithms of SEB concentration and NoV target concentration ( () is the x-axis, corresponding to The values ​​are set to the ordinate. A standard curve is plotted using linear regression fitting with Origin software. The concentrations of SEB and NoV in the sample are then calculated based on the equation of the standard curve.

[0081] Experimental results:

[0082] Sensitivity verification: SEB concentrations ranged from 0.01 to 10 ng / mL. and They exhibit a good linear relationship, and the linear equation is: Detection limit ( )for NoV target in Within the concentration range, and They exhibit a good linear relationship, and the linear equation is: The detection limit is .

[0083] Specificity verification: In SEB detection, there was only a 38% cross-reactivity with SEA, and the cross-reactivity rate with pollutants such as AFB1, CAP, and OTC was less than 10%; In NoV nucleic acid detection, the fluorescence intensity of the perfectly matched target sequence was significantly higher than that of single-base mismatch (SM), double-base mismatch (DM), triple-base mismatch (TM), and perfect mismatch (CM) sequences.

[0084] Accuracy verification: SEB spiked recovery experiments were performed on samples of pickled fish and braised lion's head meatballs, with recovery rates of 84%–119%; NoV nucleic acid spiked recovery experiments were performed on 10% bovine serum solution, with recovery rates of 95%–102%; the relative standard deviation (RSD) of all results was less than 10%.

[0085] The above embodiments can be implemented in whole or in part by software, hardware, firmware or other arbitrary combinations. When implemented by software, the above embodiments can be implemented in whole or in part in the form of a computer program product.

[0086] Those skilled in the art will recognize that the modules and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0087] In addition, the functional modules in the various embodiments of this application can be integrated into one processing module, or each module can exist physically separately, or two or more modules can be integrated into one module.

[0088] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0089] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-analyte fluorescent immunoassay method based on TMSD-DNAzyme, characterized in that, A method for simultaneous detection of Staphylococcus aureus enterotoxin B (SEB) and GII type norovirus (NoV) comprises the following steps: S1, coating SEB capture antibody on a 96-well black microplate and blocking; S2, adding diluted immune probe and sample to be tested to form a sandwich structure through immune reaction, wherein the immune probe is a SEB detection antibody coupled with a gold nanoparticle surface and a Trigger chain (sequence: SH-TTT TTT TTT TTT TTT ATG AAGATG GAG CTG GAG TG); S3, adding nucleic acid sample to be tested and functional nucleic acid mixture to realize TMSD activation DNAzyme cutting and CHA cascade amplification through 37℃ incubation in the dark, wherein the functional nucleic acid mixture comprises P1-P3 recognition sequences, HP-A / HP-B DNAzyme substrates, and HP1-HP6 CHA hairpin structures; S4, detecting fluorescence intensity at 490nm / 520nm (FAM channel) and 532nm / 568nm (Cy3 channel) through a microplate reader to realize quantitative analysis of SEB and NoV.

2. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme according to claim 1, wherein, The P1 sequence is 5'-TCA CTC GAC GCG GTC GAA TAG TGT GTC GCT C-3', the P2 sequence is 5'-AAG TACGTC TCC GAG CCC ATC TTC AT-3', and the P3 sequence is 5'-CAC TCC AGC TCG GTC GAA CGT TGAACCCAT A-3'.

3. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme according to claim 1, wherein, The HP-A sequence is 5'-CTG CAT CCT TTT TTG AGC GAC ACA CTA T / rA / G GAC GTA CTT TTT TGGATG CAG-3', and the HP-B sequence is 5'-ATG GGT CTC ACT ATG GGT TCAACG T / rA / G GAC GTA CTT-3'.

4. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The HP1 sequence is 5'-FAM-TAT AGT GTG TCG CTC AAAAAA GGA TGC TCC AAT CAC AAC TGC ATCCTT TTT TGA GCG ACA-BHQ1-3', and the HP4 sequence is 5'-Cy3-CGT TGAACC CAT AGT GAG ACC CATATG GGT CAA GAC ATG GGT CTC ACT-BHQ2-3'.

5. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, In the functional nucleic acid mixture, the concentrations of P1 and P2 are both 1000 nmol / L, the concentration of P3 is 800 nmol / L, the concentration of HP-A is 1200 nmol / L, the concentration of HP-B is 1000 nmol / L, and the concentrations of HP1-HP6 are all 500 nmol / L.

6. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The preparation of the immunoprobes includes gold nanoparticle synthesis, co-coupling of SEB detection antibody and Trigger chain with a final concentration of 18 μg / mL, PEG20000 and BSA blocking, and centrifugal purification steps.

7. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The immunoreaction condition is incubation at 37℃ for 1 hour, and the signal amplification reaction condition is incubation at 37℃ for 2 hours in the dark.

8. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The coating buffer is 0.05M carbonate buffer with pH 9.6, and the reaction buffer is pH 8.0 buffer containing 10 mmol / L Tris-HCl, 50 mmol / L KCl, and 5 mmol / L MgCl2.

9. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The linear range of the SEB detection is 0.01-10 ng / mL, and the linear range of the NoV nucleic acid detection is 5-250 nmol / L.

10. The multi-analyte fluorescent immunoassay based on TMSD-DNAzyme of claim 1, wherein, The immunoprobes are diluted by 0.01M PBS at a volume ratio of 1:20 before use, and HP-A to HP6 are heated at 95℃ for 10 minutes and then naturally cooled to room temperature before use.

Images