CN113063936A

CN113063936A - Dual-signal amplification mycotoxin detection method and detection kit

Info

Publication number: CN113063936A
Application number: CN202110360353.2A
Authority: CN
Inventors: 吴薇; 姚锡京; 俞鑫; 杨庆利
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-02
Anticipated expiration: 2041-04-02
Also published as: CN113063936B

Abstract

The invention discloses a mycotoxin detection method and a detection kit with double signal amplification, and belongs to the technical field of mycotoxin detection. The mycotoxin detection method with double signal amplification combines nicking enzyme signal amplification and CRISPR/Cas12a technology, and realizes two times of signal amplification in mycotoxin detection; the mycotoxin detection method with double signal amplification is simple and convenient to operate, complex pretreatment of a sample to be detected is not needed, and the detection cost is low; the method can be used for detecting the mycotoxin with low concentration, and has high sensitivity; can be used for specific detection of mycotoxin in food, and realizes qualitative and quantitative analysis of target mycotoxin.

Description

Dual-signal amplification mycotoxin detection method and detection kit

Technical Field

The invention belongs to the technical field of mycotoxin detection, and particularly relates to a mycotoxin detection method and a detection kit with double signal amplification.

Background

At present, more than 400 types of known mycotoxins can pollute almost all crops, food is polluted by the mycotoxins, so that the food is not only rotten and deteriorated, but also the serious food poisons human bodies, and the health and life safety of the human bodies are threatened.

Mycotoxins have carcinogenic, toxic and teratogenic hazards, and can cause kidney poisoning, liver poisoning and reproductive abnormalities of people and animals. Some food-borne mycotoxins can cause acute episodes and can rapidly develop severe disease symptoms after ingestion of food products contaminated with mycotoxins. Other mycotoxins can have long-term health effects, including the induction of cancer and immunodeficiency.

In the existing detection methods, the application range of the chromatography and the immunoassay method is wide and the immunoassay method has better accuracy, wherein the immunoassay method based on the antigen-antibody reaction has the advantages of high sensitivity, good specificity, rapidness, stability, lower cost and rapid development in the field of fungus detection. However, the commonly used imported kit is expensive, so that the detection cost is too high, and the popularization and application are difficult. Due to the characteristics of wide pollution range and great harm of food-borne pathogenic microorganisms, the development of a method which is efficient, sensitive, accurate, economical and suitable for on-site rapid detection is urgently needed in contemporary society so as to reduce or avoid the harm of pathogenic microorganisms to human bodies. And an effective food-borne pathogenic microorganism and mycotoxin related detection technology, risk assessment and control technology are developed, so that the food safety and health of the nation are guaranteed.

Disclosure of Invention

The first purpose of the invention is to provide a mycotoxin detection method with double signal amplification, which comprises the following steps:

(1) adding a mycotoxin capture probe S1-Z1-MB into a sample solution to be detected for mixed reaction, so that mycotoxin in the sample to be detected is combined with a corresponding aptamer on S1-Z1-MB to form Z1-MB-mycotoxin, and an S1 sequence is released; magnetically separating the reacted solution to obtain a supernatant containing S1;

the mycotoxin capture probe S1-Z1-MB comprises a magnetic microsphere MB, a nucleic acid aptamer Z1 of the mycotoxin to be detected and a single-stranded DNA S1 containing a recognition site of Nt. AlwI nicking endonuclease; wherein, the Z1 is modified on the surface of the magnetic microsphere MB; the single-stranded DNA S1 is combined with Z1 through a sequence which is complementary to Z1 according to the base complementary pairing principle; wherein the binding strength of the aptamer Z1 to the mycotoxin is higher than the complementary strength of the single-stranded DNA S1 and the aptamer Z1; when a mycotoxin is present, aptamer Z1 is able to preferentially bind to it and release free S1;

(2) adding S2-MB into the supernatant containing S1 in the step (1), wherein S1 is combined with S2 in the S2-MB through base complementation to form S1-S2-MB;

the S2-MB comprises a magnetic microsphere MB and a single-stranded DNA S2; the S2 is modified on the surface of a magnetic microsphere MB, and the S2 contains a sequence which is completely complementary with the recognition site of Nt. AlwI nicking endonuclease on S1;

(3) adding Nt. AlwI enzyme into the S1-S2-MB solution obtained in the step (2), and performing enzyme digestion reaction after uniformly mixing; the fourth base position of the Nt.AlwI nicking endonuclease recognition site of the sequence S2 is only used for cutting the single strand by the Nt.AlwI enzyme, and a single-strand DNA sequence S3 is released; s1 falls off after enzyme digestion and is combined with new S2-MB to realize the amplification of a first enzyme digestion signal and generate a large amount of DNA single chains S3; after the reaction is finished, carrying out magnetic separation on the solution; carrying out enzyme deactivation treatment on the residual supernatant;

(4) adding Cas12a-crRNA complex and R1 into the supernatant obtained in the step (3), uniformly mixing, and reacting to form a triplex complex of Cas12 a-crRNA-R1-S3; the R1 is a DNA single strand with the same length as the S3 complete complementarity;

(5) adding a fluorescent signal substrate into the solution reacted in the step (4) to serve as a substrate for trans-cleavage of Cas12a enzyme, performing secondary enzyme digestion signal amplification, and measuring fluorescence intensity before and after reaction after the reaction is completed;

(6) comparing the measured fluorescence intensity value with a standard curve, and calculating the content of mycotoxin in the sample solution to be measured;

the fluorescent signal substrate is an oligonucleotide chain with two ends respectively containing a fluorescent group and a fluorescent quenching group.

In a specific embodiment of the present invention, the mycotoxin capture probe S1-Z1-MB is prepared by the following method:

firstly, taking monodisperse streptavidin modified magnetic microspheres MB and aptamer Z1 of mycotoxin to be detected, the 5' end of which is connected with biotin molecules, mixing and dispersing the mixture in Tris-HCl buffer solution, carrying out vortex oscillation at room temperature to modify the aptamer of the mycotoxin to be detected on the surfaces of the magnetic microspheres, and carrying out magnetic separation to obtain Z1-MB;

adding S1 ssDNA into the Z1-MB obtained in the step I, carrying out reaction after reaction liquid is evenly mixed in a vortex mode to form S1-Z1-MB, and removing supernate after magnetic separation.

In a specific embodiment of the present invention, the S2-MB is prepared by the following method:

taking monodisperse streptavidin modified magnetic microspheres MB and 5' end connection biotin molecule S2ssDNA, mixing and dispersing in Tris-HCl buffer solution, and performing vortex oscillation at room temperature to modify S2ssDNA on the surfaces of the magnetic microspheres; Z2-MB was obtained after magnetic separation.

In a specific embodiment of the invention, the Cas12a-crRNA complex is co-incubated with Cas12a enzyme in a buffered solution, pH 7.9; the Cas12a-crRNA complex formed.

In a specific embodiment of the present invention, the standard curve in step (6) is prepared by the following method:

using fungal toxin solutions to be detected with different concentration gradients as standard solutions; detecting the standard solution by adopting the methods in the steps (1) to (5) to obtain the fluorescence intensity of the standard solution; and drawing a standard curve by taking the fluorescence intensity as an ordinate and the concentration of the mycotoxin as an abscissa.

The mycotoxin detection method with double signal amplification comprises the steps of replacing an aptamer Z1 of mycotoxin, and adaptively adjusting a sequence which has a base complementary pairing relation with Z1; can be used for detecting different kinds of mycotoxins, including but not limited to zearalenone, ochratoxin, fusarium toxin, variegated aspergillin, aflatoxin, fumonisin, trichothecene compounds, luteolin, patulin and the like.

The second purpose of the invention is to provide a dual-signal amplification mycotoxin detection kit, which comprises a mycotoxin capture probe S1-Z1-MB, S2-MB, Nt. AlwI enzyme, a Cas12a-crRNA complex, single-stranded DNA R1 and a fluorescent signal substrate;

the mycotoxin capture probe S1-Z1-MB comprises a magnetic microsphere MB, a nucleic acid aptamer Z1 of the mycotoxin to be detected and a single-stranded DNA S1 containing a recognition site of Nt. AlwI nicking endonuclease; wherein, the Z1 is modified on the surface of the magnetic microsphere MB; the single-stranded DNA S1 is combined with Z1 through a sequence which is complementary to Z1 according to the base complementary pairing principle;

the S2-MB comprises a magnetic microsphere MB and a single-stranded DNA S2; the S2 is modified on the surface of a magnetic microsphere MB, and the S2 contains a sequence which is completely complementary with the recognition site of Nt. AlwI nicking endonuclease on S1; the Nt.AlwI enzyme recognizes the enzyme cutting site of the combined double strand of S1 and S2, cuts S2 and releases a single-stranded DNA sequence;

the single-stranded DNA sequence which is separated after the enzyme digestion of S2 by the AlwI is completely complementary with the single-stranded DNA R1; the sequence, a Cas12a-crRNA compound and a single-stranded DNA R1 form a complete enzyme triplex compound with enzyme digestion activity, and the complete enzyme triplex compound is used for enzyme digestion of a fluorescent signal substrate to realize signal amplification;

According to the mycotoxin detection kit with double signal amplification, the aptamer Z1 of mycotoxin is replaced, and the sequence with the base complementary pairing relation with Z1 is adaptively adjusted; can be used for detecting different kinds of mycotoxins, including but not limited to zearalenone, ochratoxin, fusarium toxin, variegated aspergillin, aflatoxin, fumonisin, trichothecene compounds, luteolin, patulin and the like.

The third purpose of the invention is to provide a kit for detecting zearalenone toxin, which comprises a mycotoxin capture probe S1-Z1-MB, S2-MB, Nt.AlwI enzyme, a Cas12a-crRNA complex, a single-stranded DNA R1 and a fluorescent signal substrate;

the mycotoxin capture probe S1-Z1-MB comprises a magnetic microsphere MB, a nucleic acid aptamer Z1 of the mycotoxin to be detected and a single-stranded DNA S1; wherein, the Z1 is modified on the surface of the magnetic microsphere MB; the single-stranded DNA S1 binds to Z1 by base complementary pairing; the nucleic acid sequence of Z1 is shown as SEQ ID NO. 1; the nucleic acid sequence of the single-stranded DNA S1 is shown as SEQ ID NO. 2;

the S2-MB comprises a magnetic microsphere MB and a single-stranded DNA S2; the S2 is modified on the surface of the magnetic microsphere MB; the nucleic acid sequence of S2 is shown as SEQ ID NO. 3;

the nucleic acid sequence of the single-stranded DNA R1 is shown as SEQ ID NO. 4;

In a specific embodiment of the invention, the Cas12a-crRNA complex is a Cas12a-crRNA complex prepared from a Cas12a enzyme and crRNA; wherein the nucleic acid sequence of the crRNA is shown as SEQ ID NO. 5.

The technical scheme of the invention has the advantages

1. The mycotoxin detection method with double signal amplification is simple and convenient to operate, complex pretreatment of a sample to be detected is not needed, and the detection cost is low;

2. the detection method can effectively amplify the signal of the mycotoxin, can be used for detecting the mycotoxin with low concentration, and has high sensitivity;

3. the detection kit can be used for specific detection of mycotoxin in food, and realizes qualitative and quantitative analysis of target mycotoxin;

4. food samples are complex in composition and thus detection of mycotoxins in food is easily affected by interference with the food matrix. The invention adopts a magnetic separation technology, specifically, specific binding is generated between a coupling specific recognition molecule on a magnetic microsphere and toxin molecules existing in food, and a formed compound is separated from other impurities under the action of magnetic force so as to quickly enrich mycotoxin.

5. The invention adopts the aptamer as a specificity recognition molecule for mycotoxin, has strong specificity, high affinity for biological molecules, good stability, no immunogenicity and toxicity, and has the advantages of easy synthesis and easy storage.

6. The detection method disclosed by the invention combines CRISPR/Cas12a technology; cas12a belongs to class 2 type V CRISPR effector proteins, and is an endonuclease that binds to and cleaves at a specific site on a target DNA under the guidance of a single-stranded guide RNA. Once the Cas12a enzyme recognizes the target DNA after binding to crRNA to form a ternary complex, the complex exhibits strong "random-cutting" activity and cuts any single-stranded DNA in the system into fragments (called trans-cutting). When the enzyme cutting activity is activated, the compound can cut the added oligonucleotide single chain with two ends modified with fluorescent groups and quenching groups, so that the fluorescence in a quenching state is recovered to be normal; the accurate quantification of the target mycotoxin can be realized by detecting the fluorescence intensity.

Drawings

FIG. 1 is a schematic diagram of the detection principle of the method of the present invention;

FIG. 2 shows fluorescence intensities before and after reaction of Cas12a enzyme to cleave a substrate in trans (abscissa is detection wavelength nm, and ordinate is fluorescence intensity);

FIG. 3 shows the results of detection of mycotoxins at different concentrations (toxin concentration on the abscissa and fluorescence intensity on the ordinate);

FIG. 4 is a graph showing the results of detection of various mycotoxins.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

The principle of the technical scheme of the invention is described by taking the detection of zearalenone toxin as an example, and is shown in figure 1:

(1) modifying an aptamer Z1 (modified with biotin) of zearalenone on the surface of a streptavidin magnetic microsphere MB, and then designing a single-stranded DNA S1 containing a recognition site of Nt. AlwI nicking endonuclease; the single-stranded DNA S1 is combined with Z1 through a sequence which is complementary to Z1 according to the base complementary pairing principle; forming a mycotoxin capture probe S1-Z1-MB; when zearalenone is present, the aptamer Z1 in S1-Z1-MB binds zearalenone with high specificity and high selectivity; thereby breaking off the S1 originally bound to Z1. Removing the magnetic beads through magnetic frame separation; a supernatant containing free S1 was obtained.

(2) Modifying single-stranded DNA S2 (modified with biotin) on the surface of a streptavidin magnetic microsphere MB; the single-stranded DNA S2 contains a sequence completely complementary to S1; adding S2-MB into the supernatant obtained in the step (1); s1 is hybridized with S2 to form a double-stranded structure.

The restriction site of the AlwI incision enzyme is a 5' -GGATC-3 '/3 ' -CCTAG-5 ' sequence, and the chain of the 5' -GGATC-3 ' sequence is cut off, and the restriction site is positioned at the 3 ' end 4 bases relative to the recognition sequence. Because the Nt. AlwI enzyme only cuts the single strand at the fourth base position after 5 '-GGATC-3' only after recognizing 5 '-GGATC-3' and the complementary sequence thereof on the double-stranded DNA, under the action of Nt. AlwI, the formation of the double-stranded (S1/S2) recognition region leads to the cutting of the S2 strand by the Nt. AlwI, and a single-stranded DNA sequence S3 is released; meanwhile, the unstable combination characteristic of the oligonucleotide short chain after enzyme digestion is utilized, so that the S1 chain falls off after the enzyme digestion is finished, and is combined with the S2 chain on another S2-MB again, and a large amount of DNA single chains S3 are generated by the circulation, the first enzyme digestion signal amplification (nicking enzyme signal amplification) is triggered, and the detection sensitivity is enhanced; after the magnetic frame separation, a supernatant containing the DNA single strand S3 was obtained.

(3) Co-incubating Cas12a with crRNA in a DEPC-configured buffer solution to form a Cas12a-crRNA complex; then designing a DNA single-stranded R1 with the same length as the CRRNA single-stranded DNA based on the Cas12a enzyme and completely complementary with S3, so that the Cas12a enzyme can stimulate the trans-cleavage activity after self-assembly; adding the Cas12a-crRNA complex and R1 into the supernatant containing the DNA single strand S3 obtained in the step (2); a triplex complex of Cas12a-crRNA-R1-S3 (enzymatically active intact enzyme) was formed. Once a stable R-loop structure is formed, Cas12a will cleave off the non-target strand R1 first, and since the non-target linkage is relatively loosely bound to Cas12a, it will be easily released from Cas12a cleavage centers, and then the free RuvC active centers will bind to and cleave off the target strand. Since the length of the RNA-DNA hybrid double strand protected by Cas12a is limited to 26bp, the DNA target strand outside 26bp is in single-stranded form, and thus it is easy to bind to the cleavage center of Cas12 a. In this process, the Nuc domain of Cas12a stabilizes the formed R-loop structure by inhibiting reannealing of the target DNA double strand through binding to non-target DNA. At this time, the idle RuvC active center can perform non-specific cleavage on any single chain, thereby realizing the detection effect of fluorescence recovery.

(4) Adding oligonucleotide chains with two ends respectively containing a fluorescent group and a fluorescence quenching group into the mixed solution obtained in the step (3) to serve as fluorescence signal substrates of Cas12 a; cutting the fluorescent signal substrate under the action of Cas12a, separating a fluorescent group and a fluorescence quenching group of the fluorescent signal substrate, and recovering fluorescence; and exciting the amplification of the second enzyme digestion signal, and further enhancing the detection sensitivity of the zearalenone toxin.

(5) And (3) obtaining the concentration of the mycotoxin in the target sample to be detected by detecting the fluorescence intensity of the system after the reaction is finished and contrasting with a standard curve.

Example 1

A kit for detecting zearalenone toxin comprises a mycotoxin capture probe S1-Z1-MB, S2-MB, Nt. AlwI enzyme, Cas12a-crRNA complex, single-stranded DNA R1 and a fluorescent signal substrate;

wherein the mycotoxin capture probe S1-Z1-MB is prepared by the following method:

(1) preparation of Z1-MB:

taking 20 mu L of monodisperse streptavidin modified magnetic microspheres MB (the concentration of the magnetic microspheres is 5mg/mL, the magnetic microspheres are purchased from a pharmaceutical analysis and quality evaluation laboratory of the institute of medicine of Tianjin university, the developing center of BeisLe chromatography technology of Tianjin) and 10 mu M of zearalenone aptamer Z150 mu L of which the 5' end is connected with biotin molecules, mixing and dispersing in 500 mu L of Tris-HCl buffer solution (10 mM Tris-HCl,1 mM EDTA,1M Na Cl, 0.01-0.1% Tween-20), and carrying out vortex oscillation at room temperature for 30min to enable the nucleic acid aptamer Z1 of zearalenone to be modified on the surfaces of the magnetic microspheres MB; in the step, the quantity of the magnetic microspheres to be taken can be calculated by referring to the loading capacity of the magnetic microspheres according to the number of the biotinylated molecules; wherein the addition amount of the biotinylation molecules is 1-2 times of the loading amount of the magnetic microspheres, so that the magnetic beads can be saturated;

wherein the nucleic acid sequence of the zearalenone aptamer Z1 is as follows:

5'-CTA CCA GCT TTG AGG CTC GAT CCA GCT TAT TCA ATT ATA CCA GCT TAT TCA ATT ATA CCA GC-3'(SEQ ID NO:1)；

secondly, separating the magnetic microspheres modified with the zearalenone aptamer Z1 from the reaction system by using a magnet to obtain Z1-MB;

③ washing the Z1-MB obtained in the step (c) with 1mL of PBS buffer solution for three times, and finally dispersing the Z1-MB in 100 μ L of PBS buffer solution, namely Z1-MB solution, and storing the solution at 4 ℃ for later use.

(2) Hybridization of S1 with Z1-MB to form a capture probe for zearalenone toxin:

taking out the solution of Z1-MB prepared in the step 1, and separating the Z1-MB by adopting a magnet;

adding 50 mu L of 10 mu M S1 ssDNA into Z1-MB, wherein the buffer solution is PBS and the reaction volume is 500 mu L;

thirdly, after the reaction liquid in the second step is evenly mixed by vortex, the reaction liquid reacts for 30min in a gas bath shaker at room temperature to obtain a capture probe (S1-Z1-MB) of zearalenone toxin, supernatant fluid is discarded after magnetic separation, the supernatant fluid is washed twice and then resuspended by 100 mu L PBS, and the supernatant fluid is kept at 4 ℃ for standby.

Wherein the nucleic acid sequence of the S1 ssDNA is as follows:

5'-GCT GGT ATA ATT AGA TCC-3'(SEQ ID NO:2)；

the S2-MB is prepared by the following method:

mixing and dispersing 20 mu L of monodisperse streptavidin modified magnetic microspheres MB (the concentration of the magnetic microspheres is 5mg/mL) and 50 mu L of S2ssDNA (biotin molecule) with 10 mu M of 5' end connected with biotin molecules into 500 mu L of Tris-HCl buffer solution (10 mM Tris-HCl,1 mM EDTA,1M Na Cl, 0.01-0.1% Tween-20), and carrying out vortex oscillation at room temperature for 30min to modify S2ssDNA on the surfaces of the magnetic microspheres MB; in the step, the quantity of the magnetic microspheres to be taken can be calculated by referring to the loading capacity of the magnetic microspheres according to the number of the biotinylated molecules; wherein the addition amount of the biotinylation molecules is 1-2 times of the loading amount of the magnetic microspheres, so that the magnetic beads can be saturated;

wherein the nucleic acid sequence of the S2ssDNA is:

5'-ATA CTC GAT ATC CAG GGA TCT AAT TAT ACC AGC GCG CAG CGT GCT CAG CGA AGG GAA AGG ATG TAG G-3'(SEQ ID NO:3)；

separating the magnetic microsphere modified with S2ssDNA from the reaction system by using a magnet, and removing the supernatant to obtain S2-MB;

③ washing the obtained S2-MB three times with 1mL of PBS buffer solution, and finally dispersing the S2-MB in 100. mu.L of PBS buffer solution, namely S2-MB solution, and storing at 4 ℃ for later use.

The Cas12a-crRNA complex is prepared by the following method:

1.5mL of EP tube was used, and 0.1. mu.L of 1. mu.M Cas12a enzyme and 0.1. mu.L of designed 1. mu.M crRNA were mixed with a buffer solution (containing 50nM NaCl, 10mM Tris-HCl,10mM MgCl) in DEPC-treated water ₂100. mu.g/mL BSA), pH 7.9, in a total volume of 100. mu.L. Cas12a-crRNA complex can be formed after standing at 37 ℃ for about 10 min.

Wherein the nucleic acid sequence of the crRNA is as follows:

5'-UAA UUU CUA CUA AGU GUA GAU CCU UCG CUG AGC ACG CUG CGC-3'(SEQ ID NO:5)；

the nucleic acid sequence of the single-stranded DNA R1 is as follows:

5'-CCT ACA TCC TTT CCC TTC GCT GAG CAC GCT GCG CGC TGG TAT A-3'(SEQ ID NO:4)；

the fluorescent signal substrate is:

5'-FAM/AAA AAA AAA AAA AAA AAA AA/3'-BHQ(SEQ ID NO:6)；

all single chains in the reaction are heated at 95 ℃ for 10min before the reaction and quickly cooled to 4 ℃, and are stored at 4 ℃ for standby.

Example 2

A method for detecting zearalenone toxin with double signal amplification comprises the following steps:

(1) adding 100 mu L of the zearalenone toxin capture probe S1-Z1-MB prepared in example 1 into a sample solution to be detected, wherein the total volume is 500 mu L; placing the centrifuge tube on a rotary mixer at room temperature, carrying out rotary mixing at room temperature for 30min for reaction, fully reacting the mycotoxin with the probe, combining the mycotoxin in the sample to be detected with a corresponding nucleic acid aptamer on S1-Z1-MB to form Z1-MB-mycotoxin, and releasing an S1 sequence; magnetically separating the reacted solution to obtain a supernatant containing S1; transfer to a new EP tube at 4 ℃ until use.

(2) Adding 100 mu L (excessive) of S2-MB prepared in example 1 into the supernatant containing S1 in the step (1), uniformly mixing by vortex, reacting for 30min in a gas bath shaker at room temperature, and hybridizing S1 with S2 in S2-MB through base complementation to form a double strand to obtain S1-S2-MB complex;

(3) adding 1 unit of Nt.AlwI enzyme (added according to 5-10 units of Nt.AlwI enzyme per mu g of DNA, wherein 1 unit of Nt.AlwI enzyme is 0.1 mu L1 mu M) to the S1-S2-MB solution obtained in step (2), and the reaction buffer is 1X

Buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100. mu.g/mL BSA) reaction volume 500. mu.L. Lightly stroking and mixing evenly, and then reacting for 10min in a gas bath shaking table at room temperature. Magnetic separation, and putting the supernatant in a new EP tube, inactivating enzyme at 80 deg.C for 20 min. Rapidly cooling and storing to 4 ℃ for later use.

The fourth base position of the Nt.AlwI nicking endonuclease recognition site of the sequence S2 is only used for cutting the single strand by the Nt.AlwI enzyme, and a single-strand DNA sequence S3 is released; s1 falls off after enzyme digestion and is combined with new S2-MB to realize the amplification of a first enzyme digestion signal and generate a large amount of DNA single chains S3; the sequence of the single-stranded S3 is as follows:

5'-TAT ACC AGC GCG CAG CGT GCT CAG CGA AGG GAA AGG ATG TAG G-3'(SEQ ID NO:7)：

(4) adding the Cas12a-crRNA complex prepared in example 1 and 1 μ L of 10 μ M non-target strand R1 (wherein the mol concentration of the Cas12a-crRNA complex, R1 and single strand S3 is about 1:1:1) into the supernatant obtained in the step (3), uniformly mixing, standing at room temperature for 10min, and reacting to form a triplex complex (complete enzyme with enzyme digestion activity) of Cas12a-crRNA-R1-S3 to stimulate the trans-cleavage activity;

(5) the fluorescent signal substrate (10 μ M10 μ L) in example 1 was added to the solution reacted in step (4) to serve as a substrate for trans-cleavage of Cas12a enzyme, the total volume was adjusted to 1mL by using NEBuffer 2.1 reaction buffer diluted with DEPC water, the second enzyme digestion signal amplification was completed after reaction at 37 ℃ for about 30min, and after completion of the reaction, the fluorescence intensity before and after the reaction at the emission wavelength of 516nm was detected at the excitation wavelength of 494nm, and the results are shown in FIG. 2.

(6) Drawing a standard curve:

6 1.5mL EP tubes were each charged with 1mL of water containing no mycotoxin, and zearalenone toxin was added thereto so as to give concentrations of 1pg/mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL, and 100ng/mL, respectively.

Another 6.5 mL EP tubes were loaded with 100. mu.L of the magnetic bead suspension zearalenone toxin capture probe (S1-Z1-MB) resuspended in 100. mu. L, PBS of the above toxin at different concentration gradients, respectively, into 1.5mL EP tubes. The solution is subjected to constant volume to 500 mu L by PBS, incubated for 20 minutes at room temperature, and slowly shaken to fully combine the reaction; separating by a magnetic frame, and discarding the magnetic beads to obtain supernatant containing S1; magnetic separation of 100. mu.L of magnetic bead suspension S2-MB resuspended in PBS and supernatant removed, adding into S1 supernatant, adding Nt. AlwI enzyme, mixing and combining the solution for 10 minutes; then, carrying out secondary magnetic bead separation, discarding magnetic beads, and keeping a supernatant solution; the supernatant was added with 0.1 μ L of 1 μ M solution of Cas12a-crRNA complex, simultaneously added with 1 μ L of 10 μ M R1 strand, 10 μ L of 10 μ M fluorescence signal substrate reacted for 30min, and finally the reacted solution was scanned by a fluorescence spectrophotometer to measure fluorescence intensity at 494nm excitation wavelength of fluorescence emission spectrum, to obtain the standard curve 3.

(7) And (5) comparing the fluorescence intensity value measured in the step (5) with the standard curve in the step (6), and calculating the content of the mycotoxin in the sample solution to be measured.

The formula of the standard curve is that y is 796.68571 logC +2055.28571, R²The detection limit was 0.376pg/mL, 0.99213.

Example 3 specificity of zearalenone toxin detection method

Five kinds of aflatoxin, ochratoxin, fumonisin, T-2 toxin and vomitoxin are selected as interfering mycotoxin and zearalenone toxin to be tested simultaneously, so that the specificity of the zearalenone toxin detection method in embodiment 2 of the invention is verified, and the concentration of each toxin is 10 ng/mL. The detection method is the same as that of example 2; the specificity results are shown in figure 4: the fluorescence value shows that the value of the zearalenone toxin is obviously higher than that of other toxins, so that the zearalenone toxin detection method in embodiment 2 of the invention has high specificity on the zearalenone toxin.

EXAMPLE 4 specific sample detection example

Adding 100 mu L of corn oil sample to be detected into a 1.5mL EP tube, incubating the corn oil sample with 100 mu L of zearalenone toxin capture probe S1-Z1-MB prepared in the embodiment 1 for 20 minutes at room temperature, and slowly shaking to fully combine the reaction; separating the magnetic frame, and discarding the magnetic beads to obtain supernatant; the supernatant was added to the magnetically separated S2-MB prepared in example 1, and Nt. AlwI enzyme and 10X were added

After 50 mu L of Buffer, the volume of the constant volume solution is 500 mu L, and the mixture is mixed and combined for 10 minutes; then, carrying out secondary magnetic bead separation, discarding magnetic beads, and keeping a supernatant solution; adding 0.1 mu L of 1 mu M Cas12a-crRNA complex solution 100 mu L prepared in example 1 into the supernatant, simultaneously adding 1 mu L of 10 mu M R1 chain and 5 mu L of 10 mu M fluorescent signal substrate in example 1, fixing the volume to 1mL, reacting for 30min, finally scanning the reacted solution by a fluorescence spectrophotometer, measuring the fluorescence intensity by a fluorescence emission spectrum under an excitation wavelength of 494nm, introducing the measured fluorescence intensity into a standard curve, and calculating to obtain the zearalenone toxin concentration of 263pg/mL in the sample to be measured.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

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aaaaaaaaaa aaaaaaaaaa 20

<210> 7

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tataccagcg cgcagcgtgc tcagcgaagg gaaaggatgt agg 43

Claims

1. A mycotoxin detection method with double signal amplification is characterized by comprising the following steps:

2. The method for detecting mycotoxin with dual signal amplification of claim 1, wherein the mycotoxin capture probe S1-Z1-MB is prepared by the following method:

3. The method for detecting mycotoxin with dual signal amplification according to claim 1, wherein the S2-MB is prepared by the following method:

4. The dual signal amplification mycotoxin detection method of claim 1, wherein the Cas12a-crRNA complex is co-incubated with the Cas12a enzyme in a buffered solution at pH 7.9; the Cas12a-crRNA complex formed.

5. The dual signal amplification mycotoxin detection method of any one of claims 1-4, wherein the standard curve in step (6) is prepared by:

6. The dual-signal amplification mycotoxin detection method of claim 1, wherein the mycotoxin is any one of zearalenone, ochratoxin, fusarium toxin, variegated aspergillotoxin, aflatoxin, fumonisin, trichothecene compound, penicillium flavum and patulin.

7. A dual-signal amplification mycotoxin detection kit is characterized by comprising a mycotoxin capture probe S1-Z1-MB, S2-MB, Nt. AlwI enzyme, a Cas12a-crRNA complex, single-stranded DNA R1 and a fluorescent signal substrate;

8. The dual signal amplification mycotoxin assay kit of claim 7, wherein the mycotoxin is any one of zearalenone, ochratoxin, fusarium toxin, variegated aspergillomycin, aflatoxin, fumonisin, trichothecene compound, penicillium flavum, and patulin.

9. A kit for detecting zearalenone toxin is characterized by comprising a mycotoxin capture probe S1-Z1-MB, S2-MB, Nt. AlwI enzyme, a Cas12a-crRNA complex, single-stranded DNA R1 and a fluorescent signal substrate;

10. The kit for detecting zearalenone toxin according to claim 9, wherein the Cas12a-crRNA complex is a Cas12a-crRNA complex prepared from Cas12a enzyme and crRNA; wherein the nucleic acid sequence of the crRNA is shown as SEQ ID NO. 5.