Method for rapidly detecting algal toxin based on logic gate and detection kit

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to a method for rapidly detecting algal toxin based on a logic gate and a detection kit. According to the invention, a sensing platform for detecting algal toxin based on magnetic beads is established. Different algal toxin recognition probes and assembled hairpin probes are used for constructing an MB detection platform. The fluorescent group modified assembled hairpin probe can generate different fluorescence signals which can be used for distinguishing different algal toxins, meanwhile, a sensing system with three logic gates of AND-AND, Feedforward circling and Resource circling is constructed to provide a universal sensing strategy for intelligent diagnosis of different algal toxins, and the method simplifies the operation and reduces the cost, and can be used for detecting the algal toxins of different algal toxins and detecting the algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins of different algal toxins. The whole detection process is quick in response, the operation process can be mastered without professional training, and quick popularization and use are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a method for quickly detecting algal toxins based on logic gates and a detection kit. Background Art

[0002] Algal toxins are mainly heptapeptide toxins produced by cyanobacteria and contain 2 variable amino acids and 5 constant amino acids. They can inhibit the activity of protein phosphatases, cause liver damage and even promote tumors through consumption of contaminated food and drinking water, and pose a global threat to water quality and human health.

[0003] At present, algal toxin detection technology mainly relies on large-scale instruments such as large-scale chromatography and mass spectrometry and detection methods such as enzyme-linked immunosorbent assay, which cannot realize intelligent detection of algal toxins. Therefore, rapid, accurate and intelligent detection of algal toxins is one of the top priorities for accurately assessing water quality hazards and preventing blue algae outbreaks.

[0004] Therefore, there is an urgent need to develop simple, rapid, low-cost and easy-to-promote intelligent detection methods for algal toxins. Summary of the invention

[0005] The first aspect of the present invention is to provide a probe set for identifying algal toxins.

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

[0007] The purpose of the third aspect of the present invention is to provide the use of the probe described in the first aspect of the present invention and the reagent described in the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a kit for detecting algal toxins.

[0009] The fifth aspect of the present invention aims to provide a method for detecting algal toxins.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

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

[0012] In some embodiments of the present invention, the probe sequence that specifically recognizes the algae toxin MC-LR is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] In some embodiments of the present invention, the probe sequence that specifically recognizes the algae toxin MC-LA is shown as SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In some embodiments of the present invention, the probe sequence that specifically recognizes the algae toxin MC-YR is shown as SEQ ID NO: 5 or SEQ ID NO: 6.

[0015] In a first aspect of the present invention, a set of reagents for detecting algal toxins is provided, wherein the reagents include the probe set according to claim 1 and a signal amplification reagent.

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

[0017] In some embodiments of the present invention, the assembled hairpin probes include a probe having a sequence as shown in SEQ ID NO:7, a probe having a sequence as shown in SEQ ID NO:8, and a probe having a sequence as shown in 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 probes having a sequence as shown in SEQ ID NO: 7 have a fluorescent group and a quenching group.

[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, 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 the present invention:

[0023] The reagents described in the second aspect of the present invention include algae toxin MC-LR recognition probes H1A, H1B, H1C; algae toxin MC-LA recognition probes H2A, H2B, H2C; algae toxin MC-LA recognition probes H3A, H3B, H3C, assembled hairpin probes H4A, H4B, H5A, goose H5B, auxiliary chains S1A, S1B, S2A, S2B, S3A, S3B, S4A, S4B, wherein H4A and H4B contain fluorescent groups and fluorescent quenching groups respectively, and the fluorescent groups contained in H4A and H4B have different luminescent colors. All reagents are modified with biotin, and the relevant sequences are as follows:

[0024] H1A:

[0025] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-ACCATGT-GGGCCAGATC-3'(SEQ ID NO:1)。

[0026] H1B:

[0027] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:2)。

[0028] H1C:

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

[0030] H2A:

[0031] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-CTCTGCTACC-3'(SEQ ID NO:3)。

[0032] H2B:

[0033] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-TCATCCCTTGTCAACG-3'(SEQ ID NO:4)。

[0034] H2C:

[0035] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTTGTCAACG-3'(SEQ ID NO:2)。

[0036] H3A:

[0037] 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTTGTCAACG-3'(SEQ ID NO:5)。

[0038] H3B:

[0039] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-ACATGGT-3'(SEQ ID NO:6)。

[0040] H3C:

[0041] 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTTGTCAACG-3'(SEQ ID NO:5)。

[0042] H4A:

[0043] 5'-TCAT(BHQ)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(FAM)GA-ACATGGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:7)。

[0044] H4B:

[0045] 5'-TCAT(BHQ2)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(ROX)GA-AGAAGGA-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:7)。

[0046] H5A:

[0047] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-ACCATGT-TCATCCCTTGTCAACG-3'(SEQ ID NO:8)。

[0048] H5B:

[0049] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-TCCTTCT-TCATCCCTTGTCAACG-3'(SEQ ID NO:9)。

[0050] S1A:5'-ACACACACTC-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ IDNO:10)。

[0051] S1B:5'-ACCATGT-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:11)。

[0052] S2A:5'-ACCATGT-GAGTGTGTGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:12)。

[0053] S2B:5'-ATGCATCGAT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ IDNO:13)。

[0054] S3A:5'-ACCATGT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:14)。

[0055] S3B:5'-TCCTTCT-ACGATGCAT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO:15)。

[0056] S4A:

[0057] 5'-CGTTGACAAGGGATGA-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 16).

[0058] S4B: 5'-TCCTTCT-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 17).

[0059] In the above sequence, AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 18) is a flexible structural sequence; ACCATGT is domain 1, and ACATGGT complementary to it is domain 1*; TCATCCCTTGTCAACG (SEQ ID NO: 19) is domain 2, and CGTTGACAAGGGATGA (SEQ ID NO: 20) complementary to it is domain 2*; ATAGTCC is domain 3, and GGACTAT complementary to it is domain 3*; GGGCCAGATC is domain 4, and GATCTGGCCC complementary to it is domain 4*; CTCTGCTACC is domain 5, and GGTAGCAGAG complementary to it is domain 5*; GGTAGCAGAG is domain 6, and CTCTGCTACC complementary to it is domain 6*; GAGTGTGTGT is domain 7, and ACACACACTC complementary to it is domain 7*; GGTAGCAGAG is domain 8, and CTCTGCTACC complementary to it is domain 8*.

[0060] In the auxiliary chain, the 3' end of the hairpin structure of H4A and H4B has a protruding pivot, and the protruding pivot sequence is: AGAAGGA. The 5' end of the hairpin structure of H5A and H5B has a protruding pivot, and the protruding pivot sequence is: GGACTAT.

[0061] 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.

[0062] The 5' end of auxiliary chain S1A has two binding site sequences: ACACACACTC and ACAATTACCC.

[0063] The 5' end of auxiliary chain S1B has two binding site sequences: ACCATGT and ACAATTACCC.

[0064] The 5' end of auxiliary chain S2A has two binding site sequences: ACCATGT and GAGTGTGTGT.

[0065] The 5' end of auxiliary chain S2B has two binding site sequences: ATGCATCGAT and GGGCCAGATC.

[0066] The 5' end of auxiliary chain S3A has two binding site sequences: ACCATGT and GGGCCAGATC.

[0067] The 5' end of auxiliary chain S3B has two binding site sequences: TCCTTCT and ACGATGCAT.

[0068] The 5' end of auxiliary chain S4A has two binding site sequences: CGTTGACAAGGGATGA (SEQ ID NO: 20) and CTCTGCTACC.

[0069] The 5' end of auxiliary chain S4B has two binding site sequences: TCCTTCT and CTCTGCTACC.

[0070] Based on different purposes, the probes of the present invention have different usage combinations, including:

[0071] 1) Detection of algae toxins based on molecular logic gates of "AND-AND" cascade. H1A, H2A, H3A, H4A, and H5A were incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30 minutes to form a sensitive platform. In the presence of input A, the recognition probe H1A binds to MC-LR to release the trigger DNA1. Similarly, in the presence of input B, H2A will also release the trigger DNA2. Then in the presence of input C, H3A will release the trigger DNA3. Through the coordinated molecular hybridization reaction, the complex DNA1 / 2 / 3 will form a self-assembly of the initiator DNA to activate the assembly probes H4A and H5A, generating a high fluorescence signal, and realizing the detection of the simultaneous presence of MC-LR, MC-LA, and MC-YR. The principle diagram is shown in Figure 1 As shown in A.

[0072] 2) Detection of algae toxins based on the molecular logic gate of "Feedforward circuit". H1B, H2B, H3B, auxiliary chains S1A, S2A, S3A, S4A were co-incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30min to form the sensitive platform MB1. H4A and H5A were co-incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30min to form the sensitive platform MB2. The recognition probe H1B binds to MC-LR to release the trigger DNA1. DNA1 hybridizes with S1A and S2A to initiate the self-assembly of the assembly probes H4A and H5A, generating a high fluorescence signal. Similarly, in the presence of input B, H2B will also be released to trigger DNA2. DNA2 hybridizes with S3A to initiate the self-assembly of the assembly probes H4A and H5A, generating a high fluorescence signal. Then in the presence of input C, H3B will release the trigger DNA3. DNA3 hybridizes with S4A to inhibit the self-assembly of assembly probes H4A and H5A. Through the Feedforward circuit molecular logic gate, the feedback regulation function can be realized to achieve the detection of the absence of MC-YR in the detection system. The principle diagram is shown in Figure 2 shown.

[0073] 3) Detection of algae toxins based on molecular logic gates of “Resource allocation circuit”. H1C, H2C, H3C, auxiliary chain S1B and streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) were co-incubated for 30min to form sensitive platform MB1. H4A and H5A were co-incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30min to form sensitive platform MB2. Auxiliary chains S2B, S3B, and S4B were co-incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30min to form sensitive platform MB3. H4B and H5B were co-incubated with streptavidin-modified magnetic beads SA-MB (100μL, 1.0mg / mL) for 30min to form sensitive platform MB4. In the presence of input A, H1C releases trigger DNA1 to bind to S2B, initiate MB2-mediated self-assembly (path 1), and generate FAM fluorescence signal. In the presence of input B, the released trigger DNA2 hybridizes with S2B and S3B of MB3 to activate MB4-mediated self-assembly (path 2), generating ROX fluorescence signal. In the presence of input C, trigger DNA3 hybridizes with S4B of MB3 to activate MB4-mediated self-assembly (path 2), generating 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 realizing the detection of different combinations of three algal toxins. The principle diagram is shown as follows. Figure 3 shown.

[0074] In some embodiments of the present invention, the fluorescent group is located within 5 nucleotides of the 5' end of domain 2 of the assembled hairpin probe, and the quenching group is located within 5 nucleotides of the 3' end of domain 2*. Of course, those skilled in the art can exchange the positions of the fluorescent group and the quenching group and achieve similar fluorescence effects.

[0075] The third aspect of the present invention provides a kit for detecting algal toxins, comprising the reagent described in the second aspect of the present invention.

[0076] In some embodiments of the present invention, the kit further comprises streptavidin-labeled magnetic beads and a reaction buffer.

[0077] In some embodiments of the present invention, the reaction buffer comprises a Tris-HCl buffer solution, wherein one of the feasible formulas is: 100 mM NaCl, 5 mM MgCl2, 200 mM KCl, 1 mM EDTA, pH 7.4.

[0078] In some embodiments of the present invention, the kit may further include a negative control standard and a positive control standard.

[0079] A fourth aspect of the present invention provides a method as described in any one of (a) to (c):

[0080] (a) A method for detecting the simultaneous presence of algae toxin LR, algae toxin LA, and algae toxin-YR:

[0081] 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 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 to release the trigger DNA1. Similarly, in the presence of MC-LA, H2A will also release the trigger DNA2. Then, in the presence of MC-YR, H3A will release the trigger DNA3. Through the coordinated molecular hybridization reaction, the complex DNA1 / 2 / 3 will form a self-assembly of the initiating DNA deactivation assembly probes H4A and H5A, generating a high fluorescence signal, and realizing the detection of the simultaneous presence of MC-LR, MC-LA, and MC-YR.

[0082] (b) A method for detecting the presence of only cyanotoxin-LR and / or cyanotoxin-LA in a sample:

[0083] H1B (SEQ ID NO: 2), H2B (SEQ ID NO: 4), H3B (SEQ ID NO: 6), auxiliary chains S1A (SEQ ID NO: 10), S2A (SEQ ID NO: 12), S3A (SEQ ID NO: 14), S4A (SEQ ID NO: 16) were incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form sensitive platform MB1. H4A (SEQ ID NO: 7) and H5A (SEQ ID NO: 8) were incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form sensitive platform MB2. The recognition probe H1B binds to MC-LR to release the trigger DNA1. DNA1 hybridizes with S1A and S2A to initiate the self-assembly of assembly probes H4A and H5A, generating high fluorescence signals. Similarly, in the presence of input B, H2B will also be released to trigger 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 will release the trigger DNA3. DNA3 hybridizes with S4A to inhibit the self-assembly of assembly probes H4A and H5A. Through the Feedforwardcircuit molecular logic gate, the feedback regulation function can be realized, and the detection of MC-YR in the absence of the detection system can be realized.

[0084] (c) A method for detecting different combinations of algae toxin-LR, algae toxin-LA, and algae toxin-YR:

[0085] H1C (SEQ ID NO: 2), H2C (SEQ ID NO: 2), H3C (SEQ ID NO: 5), auxiliary chain S1B (SEQ ID NO: 11) were incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form sensitive platform MB1. H4A (SEQ ID NO: 7, labeled with FAM) and H5A (SEQ ID NO: 8) were incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form sensitive platform MB2. Auxiliary chains S2B (SEQ ID NO: 13), S3B (SEQ ID NO: 15), and S4B (SEQ ID NO: 17) were incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form sensitive platform MB3. H4B (SEQ ID NO: 7, labeled with 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 the trigger DNA1 to bind to S2B, initiates MB2-mediated self-assembly (path 1), and generates FAM fluorescence signal. In the presence of MC-LA, the released trigger DNA2 hybridizes with S2B and S3B of MB3 to activate MB4-mediated self-assembly (path 2), generating ROX fluorescence signal. In the presence of MC-YR, the trigger DNA3 hybridizes with S4B of MB3 to activate MB4-mediated self-assembly (path 2), generating ROX fluorescence signal. Through the resource allocation circuit molecular logic gate, the resource allocation of magnetic beads can be realized, thereby generating high fluorescence signals and realizing the detection of different combinations of three algal toxins.

[0086] In the above method, the final concentration of each independent probe, assembled hairpin probe, and auxiliary chain is: 0.01μM! 10μM.

[0087] The beneficial effects of the present invention are:

[0088] The present 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 produce different fluorescent signals, which can be used to distinguish different algal toxins. At the same time, the sensing system of three logic gates, "AND-AND", "Feedforward circuit" and "Resourceallocation circuit", is constructed to provide a universal sensing strategy for the intelligent diagnosis of different algal toxins. This method simplifies the operation and reduces the cost. The entire detection process responds quickly, and the operation process can be mastered without professional training, which is convenient for rapid promotion and use. The detection method and detection kit described in the present invention are of great significance for the rapid detection of algal toxins in biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0090] Figure 1 The “AND-AND” logic gate detection result of the present invention, wherein A is a principle schematic diagram, B is an input and output result diagram, and C is an actual detection result.

[0091] Figure 2 Schematic diagram of the detection principle of the logic gate of the "Feedforward circuit" of the present invention.

[0092] Figure 3 Schematic diagram of the logic gate detection principle of the "Resource allocation circuit" of the present invention.

[0093] Figure 4 It is the detection result of the logic gate of the "Feedforward circuit" of the present invention, wherein A is a flow chart, B is a schematic diagram of the input and output results, C is the actual detection result of the fluorescence intensity, and D is the actual detection result of the shooting under blue light.

[0094] Figure 5 It is the detection result of the logic gate of the "Resource allocation circuit" of the present invention, wherein A is a flow chart, B is a schematic diagram of the input and output results, C is the actual detection result of the fluorescence intensity, and D is the actual detection result of the shooting under blue light.

[0095] Figure 6 This is a specific result for the present invention.

[0096] Figure 7 The results of the present invention on the actual sample detection, wherein A corresponds to the FAM fluorescence spectrum result; B corresponds to the ROX fluorescence spectrum result.

[0097] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0098] Example 1 Probe Design

[0099] The probe sequences designed by the present invention are as follows:

[0100] Probe H1A:

[0101] 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCC ATACCACCTCATTATGCCCCATCTCCATCTCCGC-ACCATGT-GGGCCAGATC-3' (SEQ ID NO: 1).

[0102] Probe H1B:

[0103] 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCC ATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0104] Probe H1C:

[0105] 5'-Biotin-AAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCC ATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3' (SEQ ID NO: 2).

[0106] Probe H2A:

[0107] 5'-Biotin-AAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCCG-GATCTGGCCC-CTCTGCTACC-3' (SEQ ID NO: 3).

[0108] Probe H2B:

[0109] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-CACGCACAGAAGACACCTACAGGGCCAGATCACAATCGGTTAGTGAACTCGTACGGCGCG-GATCTGGCCC-TCATCCCTTGTCAACG-3'

[0110] (SEQ ID NO: 4).

[0111] Probe H2C:

[0112] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGCGCCAAACAGGACCACCATGACAATTACCC ATACCACCTCATTATGCCCCATCTCCATCTCCGC-GGGTAATTGT-TCATCCCTTGTCAACG-3'(SEQ ID NO: 2).

[0113] Probe H3A:

[0114] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG-3'(SEQ ID NO: 5).

[0115] Probe H3B:

[0116] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-ACATGGT-3'(SEQ ID NO: 6).

[0117] Probe H3C:

[0118] 5'-Biotin-AAAAAAAAAAAAAAAAAAA-GGACAACATAGGAAAAAGGCTCTGCTACCGGATCCCTGTTGTATGGGCATATCTGTTGAT-GGTAGCAGAG-TCATCCCTTGTCAACG-3'(SEQ ID NO: 5).

[0119] Probe H4A:

[0120] 5'-TCAT(BHQ)CCCTTGTCAACG-GGACTAT-CGTTGACAAGGGAT(FAM)GA-ACATGGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO: 7).

[0121] Probe H4B:

[0122] 5'-TCAT(BHQ2)CCCTTGTCAACG-ATAGTCC-CGTTGACAAGGGAT(ROX)GA-AGAAGGA-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO: 7).

[0123] Probe H5A:

[0124] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-ACCATGT-TCATCCCTTGTCAACG-3'(SEQ ID NO: 8).

[0125] Probe H5B:

[0126] 5'-Biotin-AAAAAAAAAAAAAAAAAAAA-GGACTAT-CGTTGACAAGGGATGA-TCCTTCT-TCATCCCTTGTCAACG-3'(SEQ ID NO: 9).

[0127] Probe S1A: 5'-ACACACACTC-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO: 10).

[0128] Probe S1B: 5'-ACCATGT-ACAATTACCC-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO: 11).

[0129] Probe S2A: 5'-ACCATGT-GAGTGTGTGT-AAAAAAAAAAAAAAAAAAAA-Biotin-3'(SEQ ID NO: 12).

[0130] Probe S2B: 5'-ATGCATCGAT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 13).

[0131] Probe S3A: 5'-ACCATGT-GGGCCAGATC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 14).

[0132] Probe S3B: 5'-TCCTTCT-ACGATGCAT-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 15).

[0133] Probe S4A:

[0134] 5'-CGTTGACAAGGGATGA-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 16).

[0135] Probe S4B: 5'-TCCTTCT-CTCTGCTACC-AAAAAAAAAAAAAAAAAAAAA-Biotin-3' (SEQ ID NO: 17).

[0136] Example 2 AND-AND logic gate detection schematic

[0137] 1. Experimental methods

[0138] (1) Formation of H1A, H2A, H3A, H4A, and H5A hairpin probes. 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) at room temperature and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form H1A, H2A, H3A, H4A, and H5A hairpin structures.

[0139] (2) Formation of SA-MB / H1A / / H2A / H3A / H4A / H5A complex. 100 nM H1A, 100 nM H2A, 100 nM H3A, 100 nM H4A, 200 nM H5A were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H1A / / H2A / H3A / H4A / H5 complex.

[0140] (3) AND-AND logic gate detection. After magnetic separation, the 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, the fluorescence spectrum from 500 to 750 nm was 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.

[0141] 2. Experimental results

[0142] Test results such as Figure 1 As shown in B and C, the system will produce a fluorescent signal only when MC-LR, MC-LA, and MC-YR are present at the same time, while other combinations will not produce a fluorescent response.

[0143] Example 3 Feedforward circuit logic gate detection schematic

[0144] 1. Experimental methods

[0145] (1) Formation of H1B, H2B, H3B, H4A, and H5A hairpin probes. 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) at room temperature and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature to form H1B, H2B, H3B, H4A, and H5A hairpin structures.

[0146] (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, 100 nM S4A were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H1B / / H2B / H3B / S1A / S2A / S3A / S4A complex (MB1). 100 nM H4A, 200 nM H5A were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes, so that a SA-MB / H4A / H5 complex (MB2) was formed.

[0147] (3) Feedforward circuit logic gate detection. After magnetic separation, MB1 and MB2 were redispersed in 50mMTris-HCl buffer. Then, 1nM target MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, the fluorescence spectrum from 500 to 750nm was recorded (Ex=495nm, Em=525nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0148] 2. Experimental results

[0149] The experimental results are as follows Figure 4 As shown, the Feedforward circuit logic gate system will produce a fluorescent signal only when MC-LR and / or MC-LA exist in the system and MC-YR does not exist, while other combinations will not produce a fluorescent response.

[0150] Example 4 Resource allocation circuit logic gate detection schematic

[0151] 1. Experimental methods

[0152] (1) Formation of hairpin probes of H1C, H2C, H3C, H4A, H5A, H4B, and H5B: 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) at room temperature, and heated at 90°C for 10 minutes. The mixture was then slowly cooled to room temperature, so that hairpin structures of H1C, H2C, H3C, H4A, H5A, H4B, and H5B were formed.

[0153] (2) Formation of SA-MB / H1C / / H2C / H3C / S1B, SA-MB / S2B / S3B / S4B, SA-MB / H4A / H5A, SA-MB / H4B / H5B complexes: 100 nM H1C, 100 nM H2C, 100 nM H3C, 100 nM S1B were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H1C / / H2C / H3C / S1B complex (MB1). 100 nM H4A, 200 nM H5A were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H4A / H5 complex (MB2). 100 nM S2B, 100 nM S3B, 100 nM S4B were incubated with MB for 30 minutes to form a SA-MB / S2B / S3B / S4B complex (MB3). 100 nM H4B, 200 nM H5B were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H4B / H5B complex (MB4).

[0154] (3) Resource allocation circuit logic gate detection. After magnetic separation, MB1-MB4 were redispersed in 50mM Tris-HCl buffer. Then, 1nM target MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, the fluorescence spectrum from 500 to 750nm was recorded (Ex=495nm, Em=525nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0155] 2. Experimental results

[0156] The 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 algae toxin combination in the sample can be determined based on the fluorescence situation.

[0157] Example 5 Specificity Detection

[0158] In this embodiment, path 1 in the logic gate of the Resource allocation circuit is taken as an example to test the specific recognition capability of the probe of the present invention.

[0159] 100 nM H1C, H4A, H5A, S1B were added to the buffer and incubated with streptavidin-modified magnetic beads SA-MB (100 μL, 1.0 mg / mL) for 30 min to form the sensitive platform MB. 1 nM MC-LA, MC-YR, MC-RR, UO2 were added to the MB system. 2 + ,Hg 2+ ,Cd 2+ ,Tetracycline and ochratoxin A were used as controls, the target MC-LR could produce high fluorescence signals, while the controls produced low signal responses, e.g. Figure 6 These data confirm the high selectivity of the sensitive platform MB for the targeted MC-LR combination.

[0160] Example 6 Actual Detection Effect

[0161] The present invention takes the "Resource allocation circuit" logic gate as an example to test the effect of the probe of the present invention in actual detection.

[0162] 1. Experimental methods

[0163] Actual sample testing:

[0164] 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 added to the filtered water samples for resource allocation circuit logic gate detection to verify the practicality of the logic gate.

[0165] (1) Formation of hairpin probes of H1C, H2C, H3C, H4A, H5A, H4B, and H5B. 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) at room temperature 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.

[0166] (2) Formation of SA-MB / H1C / / H2C / H3C / S1B, SA-MB / S2B / S3B / S4B, SA-MB / H4A / H5A, SA-MB / H4B / H5B complexes. 100 nM H1C, 100 nM H2C, 100 nM H3C, 100 nM S1B were incubated with SA-MB (XFB04, 100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H1C / / H2C / H3C / S1B complex (MB1). 100 nM H4A, 200 nM H5A were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H4A / H5 complex (MB2). 100 nM S2B, 100 nM S3B, 100 nM S4B were incubated with MB for 30 minutes to form a SA-MB / S2B / S3B / S4B complex (MB3). 100 nM H4B, 200 nM H5B were incubated with SA-MB (100 μL, 1.0 mg / mL) for 30 minutes to form a SA-MB / H4B / H5B complex (MB4).

[0167] (3) Resource allocation circuit logic gate detection. After magnetic separation, MB1-MB4 were redispersed in 50mM Tris-HCl buffer. Then, the target MC-LR, MC-LA, and MC-YR were added to the MB system respectively. After incubation for 45 minutes, the fluorescence spectrum from 500 to 750nm was recorded (Ex=495nm, Em=525nm). Corresponding fluorescence signals were generated according to different combinations, thereby achieving the purpose of intelligent detection of algal toxins.

[0168] 2. Experimental results

[0169] The results are as follows Figure 7 As shown, A is the result of FAM fluorescence spectrum, and B is the result of ROX fluorescence spectrum. The molecular logic gate detection results of "Resourceallocation circuit" are basically consistent with the results of buffer system, indicating that the possible interference from actual samples can be ignored.

Claims

1. A probe set for identifying algal toxins, characterized in that: The probe group includes probes that specifically recognize algal toxin MC-LR, algal toxin MC-LA, and algal toxin MC-YR; The probe sequence for specifically recognizing the algae toxin MC-LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The probe sequence for specifically recognizing the algae toxin MC-LA is shown in SEQ ID NO: 3 or SEQ ID NO: 4; The probe sequence that specifically recognizes the algae toxin MC-YR is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

2. A set of reagents for detecting algal toxins, characterized in that: The reagent comprises the probe set according to claim 1 and a signal amplification reagent.

3. The reagent according to claim 2, characterized in that: The signal amplification reagent includes an assembled hairpin probe and an auxiliary strand; The assembled hairpin probes include a probe having a sequence as shown in SEQ ID NO: 7, a probe having a sequence as shown in SEQ ID NO: 8, and a probe having a sequence as 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.

4. The reagent according to claim 3, characterized in that: The assembled hairpin probe, whose sequence is shown in SEQ ID NO: 7, has a fluorescent group and a quenching group.

5. The reagent according to claim 4, characterized in that: The probe set, assembled hairpin probes and auxiliary strands have biotin modifications.

6. Use of the probe set according to claim 1 and the reagent according to any one of claims 2 to 5 in the preparation of a product for detecting algal toxins.

7. A kit for detecting algal toxins, characterized in that: The kit comprises the reagent according to any one of claims 3 to 5.

8. The kit according to claim 7, characterized in that: The kit also includes streptavidin-labeled magnetic beads and a reaction buffer.

9. The method according to any one of (a) to (c): (a) A method for detecting the simultaneous presence of algae toxin LR, algae toxin LA, and algae toxin-YR: Mixing the probes with sequences such as SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 and the assembled hairpin probes with sequences such as SEQ ID NO: 7 and SEQ ID NO: 8, annealing by heating, incubating with streptavidin-labeled magnetic beads, and adding the samples for detection; (b) A method for detecting the presence of only cyanotoxin-LR and / or cyanotoxin-LA in a sample: Mixing the probes with sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 and the auxiliary chains with sequences as shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, annealing by heating, and incubating with streptavidin-labeled magnetic beads; The assembled hairpin probes with sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8 are mixed, annealed by heating, and incubated with streptavidin-labeled magnetic beads; The magnetic beads are mixed with the sample and incubated for detection; (c) A method for detecting different combinations of algae toxin-LR, algae toxin-LA, and algae toxin-YR: Mixing the probes with sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 5 and the auxiliary chain as shown in SEQ ID NO: 11, heating and annealing, and incubating with streptavidin-labeled magnetic beads; Mixing the assembled hairpin probe with the sequence of fluorescent group 1 as shown in SEQ ID NO: 7 and the assembled hairpin probe as shown in SEQ ID NO: 8, heating and annealing, and incubating with streptavidin-labeled magnetic beads; The auxiliary chains with sequences as shown in SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17 are mixed, annealed by heating, and incubated with streptavidin-labeled magnetic beads; Mixing the assembled hairpin probe with the sequence of fluorescent group 2 as shown in SEQ ID NO: 7 and the assembled hairpin probe as shown in SEQ ID NO: 9, heating and annealing, and incubating with streptavidin-labeled magnetic beads; The above product is mixed with the sample, incubated and tested.

10. The method according to claim 9, characterized in that: In the method, the final concentration of each independent probe, assembled hairpin probe, and auxiliary chain is 0.01-10 μM.

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