CN118147284A

CN118147284A - Method and kit for fluorescence detection of ochratoxin

Info

Publication number: CN118147284A
Application number: CN202410390112.6A
Authority: CN
Inventors: 陈伟; 王师沂; 陈琦; 姚帮本; 吴倩; 陈赵然; 吴胡琦
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Filing date: 2024-04-01
Publication date: 2024-06-07

Abstract

The invention discloses a method and a kit for fluorescence detection of ochratoxin. The method comprises the following steps: partially hybridizing and complementing ochratoxin aptamer and a single-stranded DNA primer to obtain a first liquid-phase reaction system containing an aptamer-primer recognition module; adding ochratoxin into the first liquid-phase reaction system to at least enable the ochratoxin to be specifically combined with an ochratoxin aptamer, so as to obtain a second liquid-phase reaction system containing single-stranded DNA primers; constructing a circulating strand displacement amplification reaction system by using the second liquid phase reaction system and performing a circulating strand displacement amplification reaction to obtain a third liquid phase reaction system containing deoxyribozyme; and adding the hairpin probe into the third liquid phase reaction system for reaction, thereby realizing fluorescence detection of ochratoxin. The detection method and the kit have the advantages of simple design by adopting double-signal amplification, isothermal operation process, simple fluorescence measurement model and higher sensitivity and specificity for OTA detection.

Description

Method and kit for fluorescence detection of ochratoxin

Technical Field

The invention belongs to the technical field of biochemical detection, and particularly relates to a method and a kit for detecting ochratoxin by fluorescence, in particular to a method and a kit for detecting ochratoxin by fluorescence based on isothermal cycle strand displacement and DNAzyme-mediated cycle signal response.

Background

Ochratoxin A (OTA) has a molecular formula of C ₂₀H₁₈ClNO₆ and is the most toxic of 7 ochratoxin analogs. Because of the high chemical and thermal stability of legumes, corn, coffee, milk, wine, and other foods, conventional food processing procedures cannot completely destroy or eliminate OTA in the food. Considering that the absorbability and the reversibility of OTA have strong toxic effects on human beings and animals, it is important to develop a rapid, ultrasensitive and easy-to-operate low-cost OTA accurate screening method in the aspects of food safety, environmental monitoring and risk assessment.

Currently, various high-precision, high-sensitivity and reliable chromatographic methods have been widely used as gold standard methods for quantitative OTA detection. However, the complex equipment, cumbersome sample preparation, and the need for specially trained personnel of these methods limit their widespread use in the field and high throughput screening fields. In contrast, among various recognition molecules, nucleic acid aptamers, i.e., short single-stranded oligonucleotide sequences, are used as very promising biorecognition elements for developing various biosensors. They were screened by classical aptamer index enrichment system evolution (SELEX) and synthesized in bulk in vitro. In recent years, the aptamer has the characteristics of easy chemical modification, high repeatability, high thermal stability, denaturation, hybridization reversibility and the like, which are superior to the traditional antibody, and is widely applied in different fields. However, for small molecules like OTA, the aptamer can only be attached to the target in a 1:1 stoichiometry, and the detection sensitivity is poor. Therefore, the detection method of ochratoxin, which is simple and convenient to operate, short in detection time, high in sensitivity and good in specificity, is a problem to be solved urgently.

Disclosure of Invention

The invention mainly aims to provide a method and a kit for fluorescence detection of ochratoxin, which are used for overcoming the defects of the prior art.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a method for fluorescence detection of ochratoxin, which comprises the following steps:

Partially hybridizing and complementing ochratoxin aptamer and a single-stranded DNA primer to obtain a first liquid-phase reaction system containing an aptamer-primer recognition module;

adding ochratoxin into the first liquid-phase reaction system to at least enable the ochratoxin to be specifically combined with an ochratoxin aptamer, so as to obtain a second liquid-phase reaction system containing single-stranded DNA primers;

constructing a circulating strand displacement amplification reaction system by using the second liquid phase reaction system and performing a circulating strand displacement amplification reaction to obtain a third liquid phase reaction system containing deoxyribozyme;

And adding a hairpin probe into the third liquid phase reaction system to react so as to recover a fluorescent signal, thereby realizing fluorescence detection of ochratoxin.

The embodiment of the invention also provides a kit for fluorescence detection of ochratoxin, which comprises:

an aptamer recognition module; wherein the aptamer recognition module comprises an ochratoxin aptamer and a single-stranded DNA primer;

a circulating strand displacement amplification module;

and, a fluorescent signal output module; the signal output module comprises deoxyribozyme and hairpin probes.

The embodiment of the invention also provides the application of the kit in preparing a product for fluorescence detection of ochratoxin or fluorescence detection of ochratoxin.

Compared with the prior art, the invention has the beneficial effects that: the qualitative detection limit of the fluorescence detection method is 0.001-100pg/mL. Compared with the existing instrument method or test strip method for detecting OTA, the method of the invention gets rid of the dependence on expensive instruments (such as a temperature-variable PCR instrument and the like) and precise operation, simplifies the detection means of OTA, improves the detection sensitivity, has stronger specificity and lower cost, is more convenient to operate, effectively improves the detection accuracy, and can fully meet the requirements of on-site rapid detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.

FIG. 1 is a schematic diagram of an ochratoxin A fluorescence detection method in an exemplary embodiment of the invention;

FIGS. 2 a-2 b are graphs showing the results of the feasibility of the fluorescence detection method of ochratoxin A in example 1 of the invention;

FIGS. 3 a-3 b are graphs showing the sensitivity test results of the ochratoxin A fluorescence method of example 1 of the present invention;

FIG. 4 is a graph showing the results of a specific test of the fluorescence detection method of ochratoxin A in example 1 of the present invention.

Detailed Description

In view of the defects of the prior art, the inventor of the present invention has provided the technical scheme of the present invention through long-term research and a large amount of practice, and the method is mainly based on isothermal cycle strand displacement and DNAzyme-mediated cycle signal response fluorescence detection of ochratoxin A, and the method has the advantages of short detection time, high sensitivity, good specificity, simple operation and low detection cost, and meets the requirements of on-site rapid detection.

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Specifically, as one aspect of the technical scheme of the invention, the method for fluorescence detection of ochratoxin comprises the following steps:

Constructing a circulating strand displacement amplification reaction system by using the second liquid phase reaction system and performing a circulating strand displacement amplification reaction to obtain a third liquid phase reaction system containing deoxyribozyme (DNAzyme);

In some preferred embodiments, the ochratoxin aptamer has a nucleotide sequence as set forth in SEQ ID No. 1.

In some preferred embodiments, the single stranded DNA primer has a nucleotide sequence as set forth in SEQ ID NO. 2.

Specifically, the single-stranded DNA primer may also be a nucleotide sequence shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, preferably a nucleotide sequence shown as SEQ ID NO. 1.

In some preferred embodiments, the ochratoxins include, but are not limited to, ochratoxin a.

In some preferred embodiments, the deoxyribose enzyme has a nucleotide sequence as set forth in SEQ ID NO. 3.

In some preferred embodiments, the hairpin probe has a nucleotide sequence as set forth in SEQ ID NO. 4.

In some preferred embodiments, the circulating strand displacement amplification reaction system further comprises a DNA polymerase, deoxyribotriphosphates, a nicking endonuclease, a first template probe, and a second template probe. The circulating strand displacement reaction system may also comprise other conventional components such as buffer salts, as known to those skilled in the art.

Further, the 3 'end of the first template probe is capable of complementary pairing with at least the 5' end of the single stranded DNA primer.

Further, the first template probe contains at least one enzyme cleavage site of a nicking endonuclease.

Further, the first template probe has a nucleotide sequence as shown in SEQ ID NO. 5.

Further, the nucleotide sequence of the first template probe is complementary to the nucleic acid sequence of the single stranded DNA primer in reverse order.

Further, the 3 'end of the second template probe is capable of complementary pairing with at least the 5' end of the deoxyribozyme.

Further, the second template probe contains at least one enzyme cleavage site for a nicking endonuclease.

Further, the second template probe has a nucleotide sequence as shown in SEQ ID NO. 6.

Further, the nucleotide sequence of the second template probe is complementary to the nucleic acid sequence of the deoxyribozyme in the reverse order.

In some preferred embodiments, both ends of the hairpin probe are modified with a fluorescent group and a quenching group, respectively.

In some preferred embodiments, the hairpin probe has a modified nucleotide sequence with an RNA site that can be specifically recognized and cleaved by Mg ²⁺.

In some preferred embodiments, the method of fluorescence detection of ochratoxins quantitatively detects in the range of 0.001-100pg/mL.

In some more specific embodiments, the method of fluorescence detection of ochratoxins comprises:

S1, partially hybridizing and complementing an aptamer of ochratoxin A with a single-stranded DNA primer to form a first liquid-phase reaction system containing an aptamer-primer recognition module;

S2, adding ochratoxin A into the first liquid-phase reaction system, so that the ochratoxin A and the aptamer are specifically combined, the partially complementary single-stranded DNA primer is competed and is free in the liquid-phase reaction system, and a second liquid-phase reaction system containing the single-stranded DNA primer is obtained;

s3, constructing a circulating strand displacement amplification reaction system in the second liquid phase reaction system, and then performing a circulating strand displacement amplification reaction to obtain a third liquid phase reaction system containing a large amount of DNAzyme;

S4, adding a hairpin probe which is modified with Mg ²⁺ for specific recognition and can be cut into the third liquid phase reaction system, enabling the hairpin probe to be combined with DNAzyme, enabling the hairpin probe to be cut off under the action of Mg ²⁺, enabling fluorescent signals to be recovered, and accordingly achieving fluorescent detection of ochratoxin A, wherein the hairpin probe can be complementary with part of nucleotide sequences of the DNAzyme sequences.

In one embodiment, the nucleotide sequence of the ochratoxin A aptamer is shown in SEQ ID NO. 1. The nucleotide sequence of the single-stranded DNA primer is screened, and the nucleotide sequence is shown as SEQ ID NO. 2.

In one embodiment, the circulating strand displacement amplification reaction system further comprises a DNA polymerase, a nicking endonuclease, a deoxynucleotide triphosphate, two template probes (template probe 1: the aforementioned first template probe; template probe 2: the aforementioned second template probe), and the like. The circulating strand displacement reaction system may also comprise other conventional components such as buffer salts, as known to those skilled in the art.

In one embodiment, the nucleotide sequences of the two template probes are respectively designed with an enzyme cutting site of a nicking endonuclease, and can be specifically identified under the condition that strand displacement generates double chains so as to generate enzyme cutting reaction.

Preferably, the nucleotide sequences of the two template probes 1 and 2 are shown as SEQ ID NO.5 and SEQ ID NO. 6.

Further, the nucleotide sequences of the two template probes 1 and 2 are complementary to the single-stranded DNA primer and DNAzyme, respectively, in the reverse order.

In one embodiment, the nucleotide sequence of the DNAzyme is shown in SEQ ID No. 3.

In one embodiment, the hairpin probe has a nucleotide sequence shown in SEQ ID NO. 4.

Further, the 5 'and 3' ends of the hairpin probe nucleotide sequence are respectively marked and modified with a fluorescent group and a quenching group; RNA sites which can be specifically recognized and cleaved by Mg ²⁺ are modified in the middle sequence of the hairpin probe.

In one embodiment, the ochratoxin a is isolated from contaminated cereal grains.

Referring to fig. 1, the 9 fluorescence detection method of the present invention mainly includes an amplification step and a signal detection step. Wherein the amplification step comprises two parts of an aptamer competition and a single-stranded DNA primer-initiated cyclic strand displacement amplification step.

The aptamer competition step refers to that ochratoxin A can specifically identify and bind with an aptamer, so that partial complementary strands (primers) of the aptamer are competing, and the dropped single-stranded DNA primers are dissociated in a liquid phase system.

The primer-initiated cyclic strand displacement amplification step includes a process in which strand displacement amplification and nicking endonucleases are repeatedly alternated. Specifically, the strand displacement amplification process is that the single-stranded DNA primer and the 5' end of the template 1 can be complementarily identified to the enzyme cutting site sequence of the nicking endonuclease, and the isothermal amplification is initiated under the action of the DNA polymerase. Then, under the condition that the enzyme cutting site is generated, the single-stranded product which is generated after amplification and is in complete complementary pairing with the template 1 is cut by the cutting endonuclease, and under the action of the DNA polymerase, the single-stranded product (namely DNAzyme sequence) is continuously generated by amplification; the large amount of DNAzyme products generated by amplification are dissociated in an amplification system, can be complementarily matched and combined with the 5' end of the template strand 2 to the enzyme cutting site of the nicking endonuclease, and the same amplification and nicking steps are alternately performed, so that the template strand 2 is used as the template for amplification, and a large amount of single-stranded DNA primer products are dissociated in the system, so that a new strand displacement amplification can be continuously initiated.

Wherein, the working concentration of the aspergillus ochraceus aptamer can be 50-300 nM; the working concentration of the single-stranded DNA primer can be 50-300 nM, and the volume concentration ratio of the single-stranded DNA primer to the ochratoxin aptamer can be 1:1, a step of; the working concentration of the template probe 1 can be 0.1 mu M-1 mu M; the working concentration of the template probe 2 may be 1 μm to 10 μm; the concentration of the hairpin probe is 0.2 nM-400 nM.

Wherein the annealing conditions for the aptamer to the partially complementary single stranded DNA primer may include: reacting at about 60deg.C for about 30 min; the reaction sequence of the cyclic strand displacement amplification may comprise: amplifying and cutting at about 37 ℃ for about 1 h; the reaction program of the fluorescent signal output module may include: the cutting reaction is circularly carried out for about 1h at about 37 ℃.

After the cutting reaction step is finished, detecting a cut product by using a fluorescence spectrophotometer, and obtaining a detection result of the sample to be detected according to the size of a fluorescence value, thereby realizing fluorescence detection of the sample to be detected.

Another aspect of the embodiments of the present invention also provides a kit for fluorescence detection of ochratoxin, comprising:

a circulating strand displacement amplification module;

In some preferred embodiments, the circulating strand displacement amplification module comprises a DNA polymerase, a deoxyribotriphosphate, a nicking endonuclease, a first template probe, and a second template probe. The circulating strand displacement amplification module may also comprise other conventional components such as buffer salts, as known to those skilled in the art.

In another aspect, the embodiment of the invention also provides the application of the kit in preparing a product for fluorescence detection of ochratoxin or fluorescence detection of ochratoxin.

The qualitative detection limit of the fluorescence detection method is 0.001-100pg/mL. Compared with the existing instrument method or test strip method for detecting OTA, the method of the invention gets rid of the dependence on expensive instruments (such as a temperature-variable PCR instrument and the like) and precise operation, simplifies the detection means of OTA, improves the detection sensitivity, has stronger specificity and lower cost, is more convenient to operate, effectively improves the detection accuracy, and can fully meet the requirements of on-site rapid detection.

The fluorescence detection method and the kit for ochratoxin provided by the invention have the advantages of accuracy, sensitivity and selectivity in isothermal conditions which are easy to realize; in addition, since Mg ²⁺ is already present in the buffer of the amplification system, no additional Mg ²⁺ is needed to activate DNAzyme, further highlighting the simplicity of the signal output module, the cyclic strand displacement amplification reaction and cyclic DNAzyme cleavage integration strategy not only improves the sensing performance of OTA, but also provides a new universal ultrasensitive technology platform for trace analysis of different analytes.

The technical scheme of the present invention is further described in detail below with reference to several preferred embodiments and the accompanying drawings, and the embodiments are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

The experimental materials used in the examples described below, unless otherwise specified, were all commercially available from conventional biochemicals. The nucleotide sequences of ochratoxin A aptamer (SEQ ID NO. 1), single-stranded DNA primer 1 (SEQ ID NO. 2), single-stranded DNA primer 2 (SEQ ID NO. 7), single-stranded DNA primer 3 (SEQ ID NO. 8), single-stranded DNA primer 4 (SEQ ID NO. 9), single-stranded DNA primer 5 (SEQ ID NO. 10), single-stranded DNA primer 6 (SEQ ID NO. 11), template probes 1 (SEQ ID NO. 5) and 2 (SEQ ID NO. 6), DNAzyme (SEQ ID NO. 3), hairpin probe (SEQ ID NO. 4) used therein are shown in Table 1 below.

TABLE 1 nucleotide sequences of partial reagents used in the examples of the present invention

Example 1: the fluorescence detection method of ochratoxin A comprises the following steps:

(1) Aptamer-primer complex recognition module formation:

mu.L of 200nM aptamer probe and its partially complementary pair of single stranded DNA primers (1. Mu.L 200 nM) were well mixed in binding buffer (10 mM Tris-HCl,120mM NaCl,10mM KCl,20mM MgCl ₂, 0.05%PEG 1000,pH 7.4). After 4.5. Mu.L of the aptamer-primer mixture was denatured by heating at 95℃for 5min, the temperature was reduced to 60℃to anneal the two single strands. After 30min, the aptamer-primer complex successfully formed a double-stranded recognition structure.

(2) Competing reaction of primers:

In the double-stranded structure complex liquid phase system with the coupled aptamer-primer obtained in the step (1), 3. Mu.L of OTA aqueous solution with different concentrations (1 fg/mL, 10fg/mL, 100fg/mL, 1pg/mL, 10pg/mL, 100 pg/mL) is added respectively, and the mixture is shaken at room temperature for 30min to perform primer competition reaction, thus obtaining solution A (7.5. Mu.L).

(3) Cyclic strand displacement amplification

Adding components required by a strand displacement amplification system into the solution A obtained in the step (2): mu.L of 500nM template probe 1, 2. Mu.L of 300nM template probe 2,2.5mM dNTPs, 1. Mu.L of 0.2U Klenow Fragment (3 '. Fwdarw.5' exo-), 1.5. Mu.L of 10U Nb.BbvCI and 2. Mu.L of buffer solution required for each DNase, and ddH ₂ O was added to make the system volume up to 20. Mu.L to give solution B. Solution B was incubated at 37 ℃ for 1h to generate enough DNAzyme probes for the following cycling fluorescence signal output module. Solution B was heated at 75deg.C for 20min to inactivate the enzymes in the system, thereby stopping the reaction.

Wherein the composition of the circulating strand displacement amplification reaction system is shown in Table 2 (20. Mu.L)

TABLE 2 composition of the circulating strand displacement amplification reaction system in this example

Component (A)	Dosage of
		Klenow Fragment(3’-5’exo-)	0.2U
Klenow Fragment (3 '-5' exo-) buffer	2μL
		dNTPs	2.5mM
Nb.BbvCI nicking endonucleases	0.5U
		rCutSmart	2μL
DEPC pure water	5μL
		Template probe 1	2μL
Template probe 2	2μL

(4) The cyclic fluorescence signal output module:

10. Mu.L of 1.5. Mu.M hairpin probe was added to the solution B, and DEPC water was added thereto so that the volume of the liquid phase system became 50. Mu.L. After isothermal incubation of the obtained 50 mu L system for 1h at 37 ℃, fluorescence measurement is carried out at 490nm by using a fluorescence spectrophotometer, and a characteristic peak value of FAM is obtained at 520nm, so that detection of OTA is realized.

Referring to fig. 2 a-2 b, when the sample does not contain OTA (the concentration of OTA is 0, and DEPC water is actually used as negative control group), the primer competition reaction cannot be performed, free primer cannot appear in the liquid phase system, and subsequent cycle strand displacement amplification cannot be initiated, so that the fluorescence signal at 520nm is weak and is a background signal; when OTA, especially OTA above 0.001pg/mL, exists in the sample, the primer competition reaction normally proceeds, the primer appears in the solution, the subsequent cycle strand displacement amplification is initiated, a sufficient amount of DNAzyme is generated, and then the cycle Mg ²⁺ is initiated to cut the hairpin probe, a fluorescent signal is generated, and the fluorescent signal is a positive signal.

Specifically, referring to fig. 3 a-3 b, the signal difference (F-F ₀) is a positive signal value excluding the background signal, corresponding to the fluorescence value actually obtained by OTA. The signal difference was significantly enhanced from 0 at an OTA concentration of 0.001pg/mL in the sample. The detection method of the embodiment has good sensitivity. Moreover, as the concentration of OTA in the sample increases gradually, the signal difference increases gradually, which indicates that the detection method of this embodiment is also suitable for quantitatively detecting the concentration of OTA from 0.001pg/mL to 100 pg/mL.

In addition, to test the selectivity of the detection method of this example, 3 μl of the aqueous solution of OTA in step (2) was replaced with 3 μl of 10ng/mL ochratoxin B (OTB), ochratoxin c (OTC), aflatoxin M1 (AFM 1), vomit toxin (DON) and Zearalenone (ZEN) respectively as positive controls, and 3 μl of DEPC water as negative controls, and the above samples were subjected to fluorescence signal detection, and the experimental results are shown in fig. 4. It can be seen that when the sample does not contain OTA, the fluorescence signal is a background signal value, which indicates that the detection method of the embodiment has good selectivity and specificity for OTA, and is helpful for improving the accuracy and convenience of OTA detection.

Examples 2 to 6

The fluorescence signal detection methods for ochratoxin A provided in examples 2 to 6 are all substantially the same as in example 1, and the only difference is that: the primer 1 is replaced by the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 respectively, and the sequences are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 11.

The experimental results of example 1 show that when primer 1 is used, the Tm value of the DNA double strand is 62.3℃and the OTA competition value F-F ₀ is 370.6;

The experimental results of example 2 show that when primer 2 is used, the Tm value of the DNA double strand is 60.2℃and the OTA competition value F-F ₀ is 275;

The experimental results of example 3 show that when primer 3 is used, the Tm value of the DNA double strand is 62.3℃and the OTA competition value F-F ₀ is 274.4;

The experimental results of example 4 show that when primer 4 is used, the Tm value of the DNA double strand is 66.9℃and the OTA competition value F-F ₀ is 151;

the experimental results of example 5 show that when primer 5 is used, the Tm value of the DNA double strand is 60.2℃and the OTA competition value F-F ₀ is 109.1;

The experimental results of example 6 show that when primer 6 is used, the Tm value of the DNA double strand is 52.4℃and the OTA competition value F-F ₀ is 26;

In addition, the inventors designed a series of other primers by using PRIMER PREMIER software and also conducted experiments in the manner of example 1, and the results show that when these primers are used, the visual detection effect of high sensitivity and high specificity on the OTA cannot be obtained at the same time.

In addition, the inventors have conducted experiments with other materials, process operations, and process conditions as described in this specification with reference to the foregoing examples, and have all obtained desirable results.

It should be understood that the technical solution of the present invention is not limited to the above specific embodiments, and all technical modifications made according to the technical solution of the present invention without departing from the spirit of the present invention and the scope of the claims are within the scope of the present invention.

Claims

1. A method for fluorescence detection of ochratoxins, comprising:

2. The method according to claim 1, characterized in that: the ochratoxin aptamer has a nucleotide sequence shown in SEQ ID NO. 1;

And/or, the single-stranded DNA primer has a nucleotide sequence shown as SEQ ID NO. 2;

And/or, the ochratoxins comprise ochratoxin a;

and/or, the deoxyribozyme has a nucleotide sequence shown as SEQ ID NO. 3;

and/or the hairpin probe has a nucleotide sequence shown as SEQ ID NO. 4.

3. The method of claim 1, wherein the circulating strand displacement amplification reaction system further comprises a DNA polymerase, deoxyribotriphosphates, a nicking endonuclease, a first template probe, and a second template probe.

4. A method according to claim 3, characterized in that: the 3 'end of the first template probe at least can be complementarily paired with the 5' end of the single-stranded DNA primer;

and/or, the first template probe at least comprises an enzyme cutting site of a nicking endonuclease;

And/or, the first template probe has a nucleotide sequence as shown in SEQ ID NO. 5;

And/or the nucleotide sequence of the first template probe is complementary to the nucleic acid sequence of the single stranded DNA primer in reverse order.

5. A method according to claim 3, characterized in that: the 3 'end of the second template probe can be at least complementarily paired with the 5' end of the deoxyribozyme;

and/or, the second template probe at least comprises an enzyme cutting site of a nicking endonuclease;

and/or, the second template probe has a nucleotide sequence as shown in SEQ ID NO. 6;

and/or the nucleotide sequence of the second template probe is complementary to the nucleic acid sequence of the deoxyribozyme in reverse order.

6. The method according to claim 1, characterized in that: fluorescent groups and quenching groups are respectively modified at two ends of the hairpin probe;

And/or, the hairpin probe has an RNA site which can be specifically recognized and cut by Mg ²⁺ modified in the nucleotide sequence.

7. The method according to claim 1, characterized in that: the quantitative detection range of the method for detecting ochratoxin by fluorescence is 0.001-100pg/mL.

8. A kit for fluorescence detection of ochratoxin, comprising:

a circulating strand displacement amplification module;

9. The kit of claim 8, wherein: the ochratoxin aptamer has a nucleotide sequence shown in SEQ ID NO. 1;

and/or, the deoxyribozyme has a nucleotide sequence shown as SEQ ID NO. 3;

and/or the hairpin probe has a nucleotide sequence shown as SEQ ID NO. 4;

And/or, the circulating strand displacement amplification module comprises a DNA polymerase, deoxyribose triphosphate, nicking endonuclease, a first template probe, and a second template probe; preferably, the first template probe has a nucleotide sequence as shown in SEQ ID NO. 5; the second template probe has a nucleotide sequence shown as SEQ ID NO. 6.

10. Use of the kit of claim 8 or 9 for the preparation of a product for fluorescence detection of ochratoxins or for fluorescence detection of ochratoxins.