Method and kit for rapid detection of algal toxins based on logic gates

Abstract

The present application belongs to the field of analysis and detection, and particularly relates to a method for rapidly detecting algal toxins based on logic gates and a detection kit. The present application establishes a sensing platform for detecting algal toxins based on magnetic beads. Different algal toxin recognition probes and assembled hairpin probes are used to construct the MB detection platform. The assembled hairpin probes modified by fluorescent groups produce different fluorescent signals, which can be used to distinguish different algal toxins. Meanwhile, the sensing system of three logic gates, i.e. "AND-AND", "Feedforward circuit" and "Resource allocation circuit", provides a general sensing strategy for intelligent diagnosis of different algal toxins. The method simplifies the operation and reduces the cost. The whole detection process is fast in response, and the operation process can be mastered without professional training, which is convenient for rapid popularization and use.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, specifically relating to a method and reagent kit for rapid detection of algal toxins based on logic gates. Background Technology

[0002] Algal toxins are primarily heptapeptide toxins produced by cyanobacteria, containing two variable amino acids and five constant amino acids. They can inhibit the activity of protein phosphatases, leading to liver damage and even tumor promotion through the consumption of contaminated food and drinking water, posing a global threat to water quality and human health.

[0003] Currently, algal toxin detection technology mainly relies on large-scale instruments such as chromatography and mass spectrometry, and detection methods such as enzyme-linked immunosorbent assay (ELISA), which cannot achieve intelligent detection of algal toxins. Therefore, rapid, accurate, and intelligent detection of algal toxins is one of the primary tasks for accurately assessing water quality hazards and preventing cyanobacterial blooms.

[0004] Therefore, there is an urgent need to develop a simple, rapid, low-cost, and easily scalable intelligent detection method for algal toxins. Summary of the Invention

[0005] The first aspect of this invention aims to provide a set of probes for identifying algal toxins.

[0006] A second aspect of the present invention is to provide a set of reagents for detecting algal toxins.

[0007] The third aspect of this invention aims to provide the application of the probe described in the first aspect of this invention and the reagent described in the second aspect of this invention.

[0008] The fourth aspect of this invention is to provide a kit for detecting algal toxins.

[0009] The fifth aspect of this invention is to provide a method for detecting algal toxins.

[0010] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0011] In a first aspect, the present invention provides a set of probes for identifying algal toxins, the probe set including probes that specifically identify algal toxins MC-LR, MC-LA, and MC-YR.

[0012] In some embodiments of the present invention, the probe sequence for specifically recognizing the algal toxin MC-LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] In some embodiments of the present invention, the probe sequence for specifically recognizing algal toxin MC-LA is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In some embodiments of the present invention, the probe sequence for specifically recognizing algal toxin MC-YR is shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0015] A second aspect of the present invention provides a set of reagents for detecting algal toxins, said reagents comprising a probe set and a signal amplification reagent.

[0016] In some embodiments of the present invention, the signal amplification reagent includes an assembled hairpin probe and an auxiliary chain.

[0017] In some embodiments of the present invention, the assembly hairpin probe includes probes with sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0018] In some embodiments of the present invention, the auxiliary chain includes probes with sequences as shown in SEQ ID NO: 10 to SEQ ID NO: 17.

[0019] In some embodiments of the present invention, the assembled hairpin probe has a fluorescent group and a quenching group as shown in SEQ ID NO: 7.

[0020] In some embodiments of the present invention, the fluorescent group is selected from any one of FAM, CY3, CY5, HTX, ROX, VIC and HEX; preferably, it is FAM or ROX.

[0021] In some embodiments of the present invention, the quenching group is selected from any one of BHQ1, BHQ2, BHQ3, and TAMRA.

[0022] The following is a detailed description of the reagent design rules of this invention:

[0023] The reagents described in the second aspect of this invention include algal toxin MC-LR recognition probes H1A, H1B, and H1C; algal toxin MC-LA recognition probes H2A, H2B, and H2C; algal toxin MC-LA recognition probes H3A, H3B, and H3C; assembly hairpin probes H4A, H4B, H5A, and H5B; and auxiliary chains S1A, S1B, S2A, S2B, S3A, S3B, S4A, and S4B. H4A and H4B each contain a fluorescent group and a fluorescence quenching group, respectively, and the fluorescent groups in H4A and H4B emit different colors. All reagents are biotin-modified, and the relevant sequences are as follows:

[0024] H1A:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-ACCATGT-GGGCCAGATC-3'(SEQ ID NO:1)。

[0025] H1B:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:2)。

[0026] H1C:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:2)。

[0027] H2A:5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-CTCTGCTACC-3'(SEQ ID NO:3)。

[0028] H2B:5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-TCATCCCTTGTCAACG-3'(SEQ ID NO:4)。

[0029] H2C:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:2)。

[0030] H3A:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTGTCAACG-3'(SEQ ID NO:5)。

[0031] H3B:5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATCTGTTGAT-GGTAGCAGAG-ACATGGT-3'(SEQ ID NO:6)。

[0032] H3C:5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATCTGTTGAT-GGTAGCAGAG-TCATCCCTGTCAACG-3'(SEQ ID NO:5)。。

[0033] H4A:5'-TCAT(BHQ)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(FAM)GA-ACATGGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:7)。

[0034] H4B:5'-TCAT(BHQ2)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(ROX)GA-AGAAGGA-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:7)。

[0035] H5A:5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-ACCATGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:8)。

[0036] H5B:5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-TCCTTCT-TCATCCCTTGTCAACG-3'(SEQ ID NO:9)。

[0037] S1A:5'-ACACACACTC-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ IDNO:10)。

[0038] S1B:5'-ACCATGT-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:11)。

[0039] S2A:5'-ACCATGT-GAGTGTGTGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:12)。

[0040] S2B:5'-ATGCATCGAT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ IDNO:13)。

[0041] S3A:5'-ACCATGT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:14)。

[0042] S3B:5'-TCCTTCT-ACGATGCAT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:15)。

[0043] S4A:5'-CGTTGACAAGGGATGA-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQID NO:16)。

[0044] S4B:5'-TCCTTCT-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:17)。

[0045] In the above sequences, AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 18) is a flexible structure sequence; ACCATGT is domain 1, and its complement ACATGGT is domain 1*; TCATCCCTTGTCAACG (SEQ ID NO: 19) is domain 2, and its complement CGTTGACAAGGGATGA (SEQ ID NO: 20) is domain 2*; ATAGTCC is domain 3, and its complement GGACTAT is domain 3*; GGGCCAGATC is domain 4, and its complement GATCTGGCCC is domain 4*; CCTTGCTACC is domain 5, and its complement GGTAGCAGAG is domain 5*; GGTAGCAGAG is domain 6, and its complement CCTTGCTACC is domain 6*; GAGTGTGTGT is domain 7, and its complement ACACACACTC is domain 7*; GGTAGCAGAG is domain 8, and its complement CCTTGCTACC is domain 8*.

[0046] In the above auxiliary chains, the 3' end of the hairpin structures H4A and H4B has a prominent fulcrum, and the prominent fulcrum sequence is: AGAAGGA. The 5' end of the hairpin structures H5A and H5B has a prominent fulcrum, and the prominent fulcrum sequence is: GGACTAT.

[0047] The H5A and H5B hairpin structures have stem-loop sequences. The stem-loop sequence of the H5A hairpin structure is: ACCATGT; the stem-loop sequence of the H5B hairpin structure is: TCCTTCT.

[0048] The 5' end of the auxiliary chain S1A has two binding site sequences: ACACACACTC and ACAATTACCC.

[0049] The 5' end of the auxiliary chain S1B has two binding site sequences: ACCATGT and ACAATTACCC.

[0050] The 5' end of the auxiliary chain S2A has two binding site sequences: ACCATGT and GAGTGTGTGT.

[0051] The 5' end of the auxiliary chain S2B has two binding site sequences: ATGCATCGAT and GGGCCAGATC.

[0052] The 5' end of the auxiliary chain S3A has two binding site sequences: ACCATGT and GGGCCAGATC.

[0053] The 5' end of the auxiliary chain S3B has two binding site sequences: TCCTTCT and ACGATGCAT.

[0054] The 5' end of the auxiliary chain S4A has two binding site sequences: CGTTGACAAGGGATGA (SEQ ID NO: 20) and CTCTGCTACC.

[0055] The 5' end of the auxiliary chain S4B has two binding site sequences: TCCTTCT and CCTTGCTACC.

[0056] Depending on the purpose, the probes of the present invention can be used in different combinations, including:

[0057] 1) Algal toxin detection based on an "AND-AND" cascade molecular logic gate. H1A, H2A, H3A, H4A, and H5A are co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form a sensitive plateau. In the presence of input A, recognition probe H1A binds to MC-LR, releasing trigger DNA1. Similarly, in the presence of input B, H2A is released, triggering DNA2. Then, in the presence of input C, H3A releases trigger DNA3. Through a co-hybridization reaction, the complex DNA 1 / 2 / 3 forms a starting DNA that activates the self-assembly of assembly probes H4A and H5A, generating a high fluorescence signal, enabling the simultaneous detection of MC-LR, MC-LA, and MC-YR. A schematic diagram is shown below. Figure 1 As shown in Figure A.

[0058] 2) Algal toxin detection based on a "feedforward circuit" molecular logic gate. H1B, H2B, H3B, and auxiliary strands S1A, S2A, S3A, and S4A were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB1. H4A and H5A were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB2. Recognition probe H1B binds to MC-LR, releasing initiating DNA1. DNA1 hybridizes with S1A and S2A, initiating the self-assembly of assembly probes H4A and H5A, generating a high fluorescence signal. Similarly, in the presence of input B, H2B is also released, initiating DNA2. DNA2 hybridizes with S3A, initiating the self-assembly of assembly probes H4A and H5A, generating a high fluorescence signal. Then, in the presence of input C, H3B releases initiating DNA3. Hybridization of DNA3 with S4A inhibits the self-assembly of probes H4A and H5A. Feedforward circuit molecular logic gates enable feedback regulation, allowing detection in the absence of MC-YR. A schematic diagram is shown below. Figure 2 As shown.

[0059] 3) Algal toxin detection based on molecular logic gates of the "Resource allocation circuit". H1C, H2C, H3C, and the auxiliary chain S1B were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB1. H4A and H5A were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB2. The auxiliary chains S2B, S3B, and S4B were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB3. H4B and H5B were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB4. In the presence of input A, H1C release triggers the binding of DNA1 to S2B, initiating MB2-mediated self-assembly (path 1) and generating a FAM fluorescence signal. In the presence of input B, the released triggers the hybridization of DNA2 with the S2B and S3B of MB3, activating MB4-mediated self-assembly (path 2) and generating a ROX fluorescence signal. In the presence of input C, triggers the hybridization of DNA3 with the S4B of MB3, activating MB4-mediated self-assembly (path 2) and generating a ROX fluorescence signal. Through the resource allocation circuit molecular logic gate, the resource allocation of magnetic beads can be achieved, thereby generating a high fluorescence signal and enabling the detection of different combinations of three algal toxins. A schematic diagram is shown below. Figure 3 As shown.

[0060] In some embodiments of the present invention, the fluorescent group is located within the 5' end of domain 2 of the assembled hairpin probe, and the quenching group is located within the 3' end of domain 2*. Of course, those skilled in the art can interchange the positions of the fluorescent group and the quenching group to achieve similar fluorescence effects.

[0061] A third aspect of the present invention provides a kit for detecting algal toxins, comprising the reagents described in the second aspect of the present invention.

[0062] In some embodiments of the present invention, the kit further includes streptomycin-avidin-labeled magnetic beads and a reaction buffer.

[0063] In some embodiments of the invention, the reaction buffer comprises a Tris-HCl buffer solution. One possible formulation is: 100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4.

[0064] In some embodiments of the present invention, the kit may further include negative control standards and positive control standards.

[0065] A fourth aspect of the invention provides a method as described in any one of (a) to (c):

[0066] (a) A method for detecting the simultaneous presence of algal toxin LR, algal toxin LA, and algal toxin-YR:

[0067] H1A (SEQ ID NO: 1), H2A (SEQ ID NO: 3), H3A (SEQ ID NO: 5), H4A (SEQ ID NO: 7), and H5A (SEQ ID NO: 8) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form a sensitive platform. In the presence of MC-LR, the recognition probe H1A binds to MC-LR and releases initiating DNA1. Similarly, in the presence of MC-LA, H2A is also released, initiating DNA2. Then, in the presence of MC-YR, H3A releases initiating DNA3. Through a co-hybridization reaction, the complex DNA 1 / 2 / 3 will form initiating DNA to activate the self-assembly of assembly probes H4A and H5A, generating a high fluorescence signal, thus enabling the simultaneous detection of MC-LR, MC-LA, and MC-YR.

[0068] (b) A method for detecting only algal toxin-LR and / or algal toxin-LA in a sample:

[0069] H1B (SEQ ID NO: 2), H2B (SEQ ID NO: 4), H3B (SEQ ID NO: 6), and auxiliary strands S1A (SEQ ID NO: 10), S2A (SEQ ID NO: 12), S3A (SEQ ID NO: 14), and S4A (SEQ ID NO: 16) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB1. H4A (SEQ ID NO: 7) and H5A (SEQ ID NO: 8) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB2. Recognition probe H1B binds to MC-LR and releases initiating DNA1. DNA1 hybridizes with S1A and S2A, initiating the self-assembly of assembly probes H4A and H5A, producing a high fluorescence signal. Similarly, in the presence of input B, H2B is also released, initiating DNA2. DNA2 hybridizes with S3A to initiate the self-assembly of assembly probes H4A and H5A, generating a high fluorescence signal. Then, in the presence of input C, H3B releases initiating DNA3. DNA3 hybridizes with S4A to inhibit the self-assembly of assembly probes H4A and H5A. Through a feedforward circuit molecular logic gate, feedback regulation can be achieved, enabling detection in the absence of MC-YR in the detection system.

[0070] (c) A method for detecting different combinations of algal toxin-LR, algal toxin-LA, and algal toxin-YR:

[0071] H1C (SEQ ID NO: 2), H2C (SEQ ID NO: 2), H3C (SEQ ID NO: 5), and auxiliary chain S1B (SEQ ID NO: 11) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB1. H4A (SEQ ID NO: 7, labeled FAM) and H5A (SEQ ID NO: 8) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB2. Auxiliary chains S2B (SEQ ID NO: 13), S3B (SEQ ID NO: 15), and S4B (SEQ ID NO: 17) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB3. H4B (SEQ ID NO: 7, labeled with the fluorescent group ROX) and H5B (SEQ ID NO: 9) were co-incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB4. In the presence of MC-LR, H1C releases initiating DNA1, which binds to S2B, initiating MB2-mediated self-assembly (path 1) and generating a FAM fluorescent signal. In the presence of MC-LA, the released initiating DNA2 hybridizes with the S2B and S3B of MB3, activating MB4-mediated self-assembly (path 2) and generating a ROX fluorescent signal. In the presence of MC-YR, initiating DNA3 hybridizes with the S4B of MB3, activating MB4-mediated self-assembly (path 2) and generating a ROX fluorescent signal. Through the resource allocation circuit molecular logic gate, the resource allocation of the magnetic beads can be realized, thereby generating a high fluorescence signal and enabling the detection of different combinations of three algal toxins.

[0072] In the above method, the final concentration of each individual probe, assembled hairpin probe, and auxiliary chain is 0.01 μM to 10 μM.

[0073] The beneficial effects of this invention are:

[0074] This invention establishes a sensing platform based on magnetic beads for algal toxin detection. Different algal toxin recognition probes and assembled hairpin probes are used to construct the MB detection platform. The assembled hairpin probes, modified with fluorescent groups, generate different fluorescence signals, which can be used to distinguish different algal toxins. Simultaneously, a sensing system with three logic gates—"AND-AND," "Feedforward circuit," and "Resourceallocation circuit"—is constructed, providing a universal sensing strategy for the intelligent diagnosis of different algal toxins. This method simplifies operation and reduces costs. The entire detection process is rapid, and the operation procedure can be mastered without professional training, facilitating rapid deployment and use. The detection method and kit described in this invention are of great significance for the rapid detection of algal toxins in biological samples. Attached Figure Description

[0075] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0076] Figure 1 The results of the detection of the "AND-AND" logic gate in this invention are shown in A, which is a schematic diagram of the principle, B is a schematic diagram of the input and output results, and C is the actual detection result.

[0077] Figure 2 This is a schematic diagram of the logic gate detection principle of the "Feedforward circuit" of the present invention.

[0078] Figure 3 This is a schematic diagram of the logic gate detection principle of the "Resource allocation circuit" of the present invention.

[0079] Figure 4 The results are the detection results of the logic gate of the "Feedforward circuit" of this invention, where A is the flowchart, B is the schematic diagram of the input and output results, C is the actual detection result of fluorescence intensity, and D is the actual detection result under blue light.

[0080] Figure 5 The results are the detection results of the logic gate of the "Resource allocation circuit" of this invention, where A is a flowchart, B is a schematic diagram of the input and output results, C is the actual detected fluorescence intensity result, and D is the actual detected result under blue light.

[0081] Figure 6 This is the result of the detection specificity of the present invention.

[0082] Figure 7 The results of this invention are the detection results of actual samples, where A corresponds to the FAM fluorescence spectroscopy result; and B corresponds to the ROX fluorescence spectroscopy result. Detailed Implementation

[0083] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0084] Example 1 Probe Design

[0085] The probe sequence designed in this invention is as follows:

[0086] Probe H1A: 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-ACCATGT-GGGCCAGATC-3' (SEQ ID NO: 1).

[0087] Probe H1B: 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0088] Probe H1C: 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0089] Probe H2A: 5'-Biotin-AAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-CTCTGCTACC-3' (SEQ ID NO: 3).

[0090] Probe H2B: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-TCATCCCTTGTCAACG-3' (SEQ ID NO: 4).

[0091] Probe H2C: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0092] Probe H3A: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG-3' (SEQ ID NO: ⑤).

[0093] Probe H3B: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-ACATGGT-3' (SEQ ID NO: ⑥). Probe H3C: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG' (SEQ ID NO: ⑤).

[0095] Probe H4A: 5'-TCAT(BHQ)CCCTTGTCAACG-GGACTAT-CGTTGACAAGGGAT(FAM)GA-ACATGGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: ⑦).

[0096] It should be noted that in the original text, there is a small error in the "SEQ ID NO" in the translation of . It should be "SEQ ID NO: 5" instead of "SEQ ID NO: ⑤". The same correction applies to . Also, in the translation of , there is an extra line break at the end which should be removed. The corrected translation is as follows: Probe H2B: <5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-TCATCCCTTGTCAACG-3' (SEQ ID NO: 4).

[0091] Probe H2C: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0092] Probe H3A: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG-3' (SEQ ID NO: 5).

[0093] Probe H3B: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-ACATGGT-3' (SEQ ID NO: 6).

[0094] Probe H3C: 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG-3' (SEQ ID NO: 5).

[0095] Probe H4A: 5'-TCAT(BHQ)CCCTTGTCAACG-GGACTAT-CGTTGACAAGGGAT(FAM)GA-ACATGGT-AAAAAAAAAAAAAAAAAAAA-Biotin-③' (SEQ ID NO: 7).

[0096] Probe H4B: 5'-TCAT(BHQ2)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(ROX)GA-AGAAGGA-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 7).

[0097] Probe H5A: 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-ACCATGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 8).

[0098] Probe H5B: 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-TCCTTCT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 9).

[0099] Probe S1A: 5'-ACACACACTC-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQID NO: 10).

[0100] Probe S1B: 5'-ACCATGT-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ IDNO: 11).

[0101] Probe S2A: 5'-ACCATGT-GAGTGTGTGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ IDNO: 12).

[0102] Probe S2B: 5'-ATGCATCGAT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQID NO: 13).

[0103] Probe S3A: 5'-ACCATGT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ IDNO: 14).

[0104] Probe S3B: 5'-TCCTTCT-ACGATGCAT-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ IDNO: 15).

[0105] Probe S4A: 5'-CGTTGACAAGGGATGA-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 16).

[0106] Probe S4B: 5'-TCCTTCT-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 17).

[0107] Example 2: Schematic diagram of AND-AND logic gate detection

[0108] 1. Experimental Methods

[0109] (1) Formation of hairpin probes H1A, H2A, H3A, H4A, and H5A. At room temperature, 100 nM H1A, 100 nM H2A, 100 nM H3A, 100 nM H4A, and 200 nM H5A were mixed in a Tris-HCl buffer solution (100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4) and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form hairpin structures of H1A, H2A, H3A, H4A, and H5A.

[0110] (2) Formation of SA-MB / H1A / / H2A / H3A / H4A / H5A complex. 100 nM H1A, 100 nM H2A, 100 nM H3A, 100 nM H4A, and 200 nM H5A were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H1A / / H2A / H3A / H4A / H5 complex.

[0111] (3) AND-AND logic gate detection. After magnetic separation, MB was redispersed in 50 mM Tris-HCl buffer. Then, 1 nM of target MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, fluorescence spectra from 500 to 750 nm were recorded (Ex = 495 nm, Em = 525 nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0112] 2. Experimental Results

[0113] Test results as follows Figure 1As shown in Figures B and C, the system only produces a fluorescent signal when MC-LR, MC-LA, and MC-YR are present simultaneously, while other compositions do not produce a fluorescent reaction.

[0114] Example 3: Schematic diagram of logic gate detection in a feedforward circuit

[0115] 1. Experimental Methods

[0116] (1) Formation of hairpin probes H1B, H2B, H3B, H4A, and H5A. At room temperature, 100 nM H1B, 100 nM H2B, 100 nM H3B, 100 nM H4A, and 200 nM H5A were mixed in a Tris-HCl buffer solution (100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4) and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form hairpin structures of H1B, H2B, H3B, H4A, and H5A.

[0117] (2) Formation of SA-MB / H1B / / H2B / H3B / S1A / S2A / S3A / S4A and SA-MB / H4A / H5A complexes. 100 nM H1B, 100 nM H2B, 100 nM H3B, 100 nM S1A, 100 nM S2A, 100 nM S3A, and 100 nM S4A were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H1B / / H2B / H3B / S1A / S2A / S3A / S4A complex (MB1). 100 nM H4A, 200 nM H5A and SA-MB (XFB04, 100 μL, 1.0 mg / mL) were incubated together for 30 minutes to form the SA-MB / H4A / H5 complex (MB2).

[0118] (3) Feedforward circuit detection. After magnetic separation, MB1 and MB2 were redispersed in 50 mM Tris-HCl buffer. Then, 1 nM of the targets MC-LR, MC-LA, and MC-YR were added to the MB system. After incubation for 45 minutes, the fluorescence spectra from 500 to 750 nm were recorded (Ex = 495 nm, Em = 525 nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0119] 2. Experimental Results

[0120] Experimental results are as follows Figure 4 As shown, the Feedforward circuit logic gate system will only generate a fluorescent signal when MC-LR and / or MC-LA are present in the system and MC-YR is absent, while other combinations will not generate a fluorescent response.

[0121] Example 4: Schematic diagram of resource allocation circuit logic gate detection

[0122] 1. Experimental Methods

[0123] (1) Formation of hairpin probes H1C, H2C, H3C, H4A, H5A, H4B, and H5B: At room temperature, 100 nM H1C, 100 nM H2C, 100 nM H3C, 100 nM H4A, 200 nM H5A, 100 nM H4B, and 200 nM H5B were mixed in a Tris-HCl buffer solution (100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4) and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form hairpin structures H1C, H2C, H3C, H4A, H5A, H4B, and H5B.

[0124] (2) Formation of SA-MB / H1C / / H2C / H3C / S1B, SA-MB / S2B / S3B / S4B, SA-MB / H4A / H5A, and SA-MB / H4B / H5B complexes: 100 nM H1C, 100 nM H2C, 100 nM H3C, and 100 nM S1B were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H1C / / H2C / H3C / S1B complex (MB1). 100 nM H4A and 200 nM H5A were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H4A / H5 complex (MB2). Incubate 100 nM S2B, 100 nM S3B, and 100 nM S4B with MB for 30 minutes to form the SA-MB / S2B / S3B / S4B complex (MB3). Incubate 100 nM H4B and 200 nM H5B with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H4B / H5B complex (MB4).

[0125] (3) Resource allocation circuit logic gate detection. After magnetic separation, MB1-MB4 were redispersed in 50 mM Tris-HCl buffer. Then, 1 nM of target MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, fluorescence spectra from 500 to 750 nm were recorded (Ex = 495 nm, Em = 525 nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0126] 2. Experimental Results

[0127] Experimental results are as follows Figure 5 As shown, different combinations of MC-LR, MC-LA, and MC-YR will cause different fluorescence reaction results, and the type of algal toxin combination in the sample can be determined based on the fluorescence.

[0128] Example 5 Specificity Detection

[0129] This embodiment uses path 1 in the logic gate of the resource allocation circuit as an example to test the specific recognition capability of the probe of the present invention.

[0130] Add 100 nM of H1C, H4A, H5A, and S1B to the buffer solution and incubate with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form a sensitive plateau (MB). Add 1 nM of each of MMC-LA, MC-YR, MC-RR, and UO2 to the MB system. 2+ Hg 2+ Cd 2+ Tetracycline and ochratoxin A were used as controls. The target MC-LR produced a high fluorescence signal, while the control produced a low signal response. Figure 6 As shown in the figure. These data confirm the high selectivity of the sensitive platform MB for the target MC-LR combination.

[0131] Example 6: Actual Detection Results

[0132] This invention uses the "Resource allocation circuit" logic gate as an example to test the effectiveness of the probe of this invention in actual detection.

[0133] 1. Experimental Methods

[0134] Actual sample testing:

[0135] Water samples from Dongting Lake were filtered through a 0.22 μm membrane to remove insoluble particles. 1 nM MC-LR, MC-LA, and MC-YR were then added to the filtered water samples for testing the logic gates in a resource allocation circuit to verify their practicality.

[0136] (1) Formation of hairpin probes H1C, H2C, H3C, H4A, H5A, H4B, and H5B. At room temperature, 100 nM H1C, 100 nM H2C, 100 nM H3C, 100 nM H4A, 200 nM H5A, 100 nM H4B, and 200 nM H5B were mixed in a Tris-HCl buffer solution (100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4) and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form hairpin structures of H1C, H2C, H3C, H4A, H5A, H4B, and H5B.

[0137] (2) Formation of SA-MB / H1C / / H2C / H3C / S1B, SA-MB / S2B / S3B / S4B, SA-MB / H4A / H5A, and SA-MB / H4B / H5B complexes. 100 nM H1C, 100 nM H2C, 100 nM H3C, and 100 nM S1B were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H1C / / H2C / H3C / S1B complex (MB1). 100 nM H4A and 200 nM H5A were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H4A / H5 complex (MB2). Incubate 100 nM S2B, 100 nM S3B, and 100 nM S4B with MB for 30 minutes to form the SA-MB / S2B / S3B / S4B complex (MB3). Incubate 100 nM H4B and 200 nM H5B with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form the SA-MB / H4B / H5B complex (MB4).

[0138] (3) Resource allocation circuit logic gate detection. After magnetic separation, MB1-MB4 were redispersed in 50 mM Tris-HCl buffer. Then, the targets MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, the fluorescence spectra from 500 to 750 nm were recorded (Ex = 495 nm, Em = 525 nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0139] 2. Experimental Results

[0140] The results are as follows Figure 7 As shown, A represents the FAM fluorescence spectroscopy results, and B represents the ROX fluorescence spectroscopy results. The molecular logic gate detection results of the "Resourceallocation circuit" are basically consistent with the results of the buffer system, indicating that the possible interference from the actual sample can be ignored.

Claims

1. A set of reagents for detecting algal toxins, characterized in that: The reagents include probe sets and signal amplification reagents; The probe set includes probes that specifically recognize algal toxins MC-LR, MC-LA, and MC-YR. The probe sequence that specifically recognizes the algal toxin MC-LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The probe sequence that specifically recognizes the algal toxin MC-LA is shown in SEQ ID NO: 3 or SEQ ID NO: 4; The probe sequence that specifically recognizes the algal toxin MC-YR is shown in SEQ ID NO: 5 or SEQ ID NO: 6; The signal amplification reagent includes an assembled hairpin probe and an auxiliary chain; The assembled hairpin probe includes a probe with the sequence shown in SEQ ID NO: 7, a probe with the sequence shown in SEQ ID NO: 8, and a probe with the sequence shown in SEQ ID NO: 9; The auxiliary chain includes probes with sequences as shown in SEQ ID NO: 10 to SEQ ID NO: 17; The assembled hairpin probe has a fluorescent group and a quenching group, as shown in SEQ ID NO:

7.

2. The reagent according to claim 1, characterized in that: The probe set, the assembled hairpin probe, and the auxiliary chain are biotin-modified.

3. The use of the reagent according to any one of claims 1 to 2 in the preparation of products for detecting algal toxins.

4. A kit for detecting algal toxins, characterized in that: The kit comprises the reagents described in any one of claims 1 to 2.

5. The reagent kit according to claim 4, characterized in that: The kit also includes streptomycin-avidin-labeled magnetic beads and a reaction buffer.

6. A method for detecting the simultaneous presence of algal toxin LR, algal toxin LA, and algal toxin-YR, comprising the following steps: The three probes with sequences as shown in SEQ ID NO: 1, 3, and 5 and the two assembled hairpin probes with sequences as shown in SEQ ID NO: 7 and 8 were mixed, heated and annealed, incubated with streptomycin-avidin-labeled magnetic beads, and then added to the sample for detection. The assembled hairpin probe has a fluorescent group and a quenching group, as shown in SEQ ID NO:

7.

7. A method for detecting only algal toxin-LR and / or algal toxin-LA in a sample, comprising the following steps: The three probes with sequences as shown in SEQ ID NO: 2, 4, and 6, and the four auxiliary strands with sequences as shown in SEQ ID NO: 10, 12, 14, and 16 were mixed, heated and annealed, and then incubated with streptomycin-labeled magnetic beads. The two assembled hairpin probes with sequences as shown in SEQ ID NO: 7 and 8 were mixed, heated and annealed, and then incubated with streptoavidin-labeled magnetic beads. The magnetic beads were mixed with the sample and incubated for testing. The assembled hairpin probe has a fluorescent group and a quenching group, as shown in SEQ ID NO:

7.

8. A method for detecting different combinations of algal toxin-LR, algal toxin-LA, and algal toxin-YR, comprising the following steps: The two probes with sequences as shown in SEQ ID NO: 2 and 5 and the auxiliary strand shown in SEQ ID NO: 11 were mixed, heated and annealed, and then incubated with streptovisitin-labeled magnetic beads. The assembled hairpin probes with the sequence of fluorescent group 1 as shown in SEQ ID NO: 7 and the assembled hairpin probes as shown in SEQ ID NO: 8 were mixed, heated and annealed, and then incubated with streptomycin-labeled magnetic beads. The three auxiliary strands with sequences as shown in SEQ ID NO: 13, 15, and 17 were mixed, heated and annealed, and then incubated with streptomycin-labeled magnetic beads. The assembled hairpin probes with the sequence of fluorescent group 2 as shown in SEQ ID NO: 7 and the assembled hairpin probes as shown in SEQ ID NO: 9 were mixed, heated and annealed, and then incubated with streptomycin-labeled magnetic beads. The above product was mixed with the sample and incubated for testing.

9. The method according to any one of claims 6 to 8, characterized in that: In the method, the final concentration of each independent probe, assembled hairpin probe, and auxiliary chain is 0.01~10μM.

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