A method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification

Abstract

The invention belongs to the field of mycotoxin detection, and specifically relates to a method for simultaneously detecting ochratoxin and aflatoxin B1 based on upconversion fluorescence cyclic amplification; the steps are: firstly preparing upconversion nanomaterials, then modifying the upconversion nanomaterials, and simultaneously preparing AuNRs solutions with specific emission wavelengths, and finally constructing OTA and AFB1 specific detection systems, and establishing a prediction model according to fluorescence spectrum information and concentration; by detecting the fluorescence spectrum information of a sample test liquid, simultaneous detection of AFB1 and OTA in the sample can be achieved; the invention can simultaneously detect two target objects, is easy to operate, and has high sensitivity and high selectivity.

Description

Technical Field

[0001] The invention belongs to the field of mycotoxin detection, and in particular relates to a method for simultaneously detecting ochratoxin (OTA) and aflatoxin B1 (AFB1) based on up-conversion fluorescence cyclic amplification. Background Art

[0002] Mycotoxins are secondary metabolites naturally produced by fungi during their growth. About 25% of food crops worldwide are infected to varying degrees. They enter the food chain mainly by contaminating the food people eat, thus causing serious impacts on human life and health. Mycotoxins often damage organs such as the liver, kidneys, and brain nervous system, causing symptoms of cirrhosis, liver cancer, acute and chronic nephritis, and bleeding in the central nervous system of the brain. Aflatoxin is the most toxic and harmful of all mycotoxins, and is therefore identified as a Class I carcinogen by the International Agency for Research on Cancer. Ochratoxin is the most widely distributed in nature and is closely related to human life. It is often found in various foods, feeds, and other agricultural and sideline products, posing a huge threat to human health. Therefore, it is crucial to establish a scientific and feasible method for detecting mycotoxins.

[0003] At present, the main methods for mycotoxin detection include thin layer chromatography, high performance liquid chromatography, high performance liquid phase mass spectrometry and immunoassay based on immune recognition. The thin layer chromatography method has complicated operation steps, low sensitivity and specificity, and can no longer meet the needs of modern detection. The commonly used chromatographic analysis method has complex and cumbersome sample pretreatment methods, and requires proficiency in instrument operation techniques, which is inconvenient for grassroots promotion. Although the immunoassay has the advantages of high sensitivity and strong specificity, it can only detect a single test, has poor repeatability and is expensive. Therefore, it is necessary to develop a method that is fast, convenient, highly sensitive, highly specific, low cost and can detect simultaneously. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of existing detection technologies and provide a method for simultaneously detecting OTA and AFB1 based on upconversion nanomaterials and RNase-H, which can eliminate the interference of other analogs and achieve low-cost, high-sensitivity, rapid and simultaneous detection.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] Step 1: Preparation of upconversion nanomaterials:

[0007] (a) Dissolving yttrium oxide, ytterbium oxide and erbium oxide in trifluoroacetic acid respectively, and then adding sodium fluoride to obtain a mixed solution A, transferring the mixed solution A to a solution containing oleylamine and 1-octadecene; continuously filling with nitrogen and performing magnetic stirring, heating the reaction and cooling to room temperature; then adding a mixed solution of cyclohexane and ethanol for centrifugal washing; the precipitate obtained by centrifugation is then washed by centrifugation with pure water, and finally freeze-dried to obtain an upconversion nanomaterial, which is recorded as UCNPs1;

[0008] (b) The method and parameter conditions are the same as step (a), except that the yttrium oxide, ytterbium oxide and erbium oxide in step (a) are replaced by yttrium oxide, ytterbium oxide and thulium oxide; and finally an upconversion nanomaterial is obtained, which is recorded as UCNPs2;

[0009] Step 2: Modification of upconversion nanomaterials:

[0010] (a) The UCNPs1 obtained in step 1 was dissolved in isopropanol A, and then distilled water and ammonia water were added in sequence, the mixture was sealed and stirred rapidly, and then a mixed solution of isopropanol B and tetraethyl orthosilicate was added dropwise, and stirring was continued for a period of time, and then isopropanol C and 3-aminopropyltriethoxysilane were added dropwise; stirring was stopped after the addition, and the mixture was allowed to stand at room temperature for aging, and then centrifuged after aging, and then freeze-dried to obtain upconversion nanoparticles with amino groups modified on the surface, which were recorded as NH 4 -UCNPs1;

[0011] (b) The operation and parameter conditions of step (b) are the same as those of step (a), except that UCNPs1 is replaced by UCNPs2; the final upconversion nanoparticles with amino groups modified on the surface are obtained, which are denoted as NH 4 -UCNPs2;

[0012] Step 3: Preparation of AuNRs with specific emission wavelength:

[0013] (a) Preparation of seed solution: chloroauric acid solution (HAuCl 4 ·4H 2 O) and cetyltrimethylammonium bromide solution (CTAB), then add sodium borohydride solution, stir for a period of time and let stand at a certain temperature to obtain seed solution, which is stored in the dark;

[0014] (b) Preparation of AuNRs solution: CTAB and sodium oleate were dissolved in deionized water A, and then silver nitrate was added. After standing for reaction, a mixed solution A was obtained. HAuCl was added to the mixed solution A. 4 ·4H 2O and stirred until colorless, then added concentrated hydrochloric acid and ascorbic acid solution in sequence, stirred to obtain a mixed solution B; the seed solution prepared in step (a) was added to the mixed solution B, stirred and then allowed to stand and centrifuged, and the obtained precipitated product was redissolved in deionized water B to obtain an AuNRs solution with a specific emission wavelength, which was stored in the dark;

[0015] Step 4: Construction of specific detection system:

[0016] (a) The NH obtained in step 2 4 -UCNPs1 was added to PBS buffer A and dissolved by ultrasonication to obtain NH 4 -UCNPs1 solution, glutaraldehyde was added, and the mixture was placed on a shaker for reaction. The precipitate after centrifugation was re-dispersed in PBS buffer B by ultrasonication, and then avidin was added and incubated by shaking. After incubation, the freeze-dried product was re-dispersed in PBS buffer C to obtain a mixed solution A. Finally, the RNA chain (RNA1) corresponding to the analyte (OTA) was added and incubated overnight. The precipitate obtained after centrifugation was re-dispersed in PBS buffer D. The obtained mixed solution was recorded as NH 4 -UCNPs1-RNA1;

[0017] (b) The operation and parameter conditions are the same as those in step (a), except that NH 4 -UCNPs1 replaced by NH 4 -UCNPs2, RNA1 is replaced by RNA2 corresponding to AFB1, and the final product is NH 4 -UCNPs2-RNA2.

[0018] (c) adding the AuNRs solution prepared in step 3 to NH4-UCNPs1-RNA1 and NH4-UCNPs1-RNA2, respectively, to obtain mixed solutions A and B, both of which are placed in a shaker for incubation, washed with PBS buffer after incubation, and centrifuged after washing to obtain products, respectively recorded as product A and product B; then, product A and product B are dispersed in PBS buffer solution to obtain two fluorescent detection probe solutions, respectively recorded as Probe-RNA1 and Probe-RNA2;

[0019] (d) selecting two base complementary pairs of DNA1-cDNA1 and DNA2-cDNA2 and mixing them with a certain concentration of OTA solution and AFB1 solution; then adding Probe-RNA1 and Probe-RNA2 obtained in step (c); finally adding RNase-H for co-incubation, and obtaining a test solution after incubation, that is, constructing an OTA and AFB1 specific detection system;

[0020] Step 5: OTA and AFB1 detection:

[0021] Taking the test solution prepared in step 4, setting the concentrations of OTA and AFB1 therein in a gradient, collecting fluorescence spectrum information of the test solution with known OTA and AFB1 concentrations, and establishing a prediction model based on the fluorescence spectrum information and concentration;

[0022] Step 6: Detection of OTA and AFB1 in samples:

[0023] Referring to the methods of GB 5009.96-2016 and GB 5009.22-2016, the sample is processed to prepare a sample extract; then, the sample extract is added to Probe-RNA1 and Probe-RNA2, and then RNase-H is added for co-incubation to obtain a sample test solution, and the fluorescence spectrum of the sample test solution is collected; then, the collected fluorescence spectrum information is imported into the prediction model established in step five; the specific concentration values ​​of OTA and AFB1 in the sample are obtained through model calculation, thereby realizing the simultaneous detection of OTA and AFB1 in the sample.

[0024] Preferably, in step 1 (a), the amount of yttrium oxide, ytterbium oxide, erbium oxide and trifluoroacetic acid is 74-80 mol: 18-24 mol: 1.8-2.2 mol: 5 mL; the concentration of sodium fluoride in the mixed solution A is 10 mmol / L;

[0025] The dosage ratio of trifluoroacetic acid to oleylamine and 1-octadecene in the mixed solution A is 5 mL:20 mL:20 mL; the dosage ratio of the mixed solution of cyclohexane and ethanol to the trifluoroacetic acid in the mixed solution A is 100-200 mL:5 mL; and the volume ratio of cyclohexane and ethanol is 2:1.

[0026] Preferably, in step 1 (a), the heating reaction temperature is 320° C. and the reaction time is 1 h.

[0027] Preferably, in step 2 (a), the dosage ratio of UCNPs1, isopropanol A, distilled water, and ammonia water is 20 mg: 50-65 mL: 15-25 mL: 2-3 mL; the dosage ratio of ammonia water to isopropanol B, tetraethyl orthosilicate, isopropanol C, and 3-aminopropyltriethoxysilane is 2-3 mL: 17-22 mL: 50-70 μL: 25-33 mL: 180-200 μL; the continuous stirring period is 3 hours; the standing aging time at room temperature is 2 hours. Among them, isopropanol A, isopropanol B, and isopropanol C are all isopropanol, and different letters are only used to distinguish the names.

[0028] Preferably, in step three (a), the dosage ratio of the chloroauric acid solution, the hexadecyltrimethylammonium bromide solution and the sodium borohydride solution is 5 mL:5 mL:0.6 mL; the concentration of the chloroauric acid solution is 0.5 mmol / L, the concentration of the hexadecyltrimethylammonium bromide solution is 0.2 mol / L, and the concentration of the sodium borohydride solution is 10 mmol / L.

[0029] Preferably, in step 3 (a), the stirring period is 2 to 5 minutes; the temperature of standing under the certain temperature condition is 30° C. and the time is 30 minutes.

[0030] Preferably, in step 3 (b), the ratio of CTAB, sodium oleate, deionized water A and silver nitrate is 1.8 g: 0.2468 g: 50 mL: 4.8 mL; the concentration of silver nitrate is 4 mmol / L; the conditions for adding silver nitrate and standing for reaction are: temperature 30° C., reaction time 15 min;

[0031] Preferably, in step 3 (b), the deionized water A in the mixed solution A and HAuCl 4 ·4H 2 O, concentrated hydrochloric acid, ascorbic acid solution dosage ratio is 50mL:50mL:0.15-0.20mL:0.15-0.20mL; the HAuCl 4 ·4H 2 The concentration of O is 1 nmol / L, and the concentration of the ascorbic acid solution is 0.064 mol / L; the stirring time after adding concentrated hydrochloric acid and ascorbic acid is 30 to 40 seconds;

[0032] Preferably, in step 3 (b), the ratio of ascorbic acid solution, seed solution and deionized water B in the mixed solution B is 0.15-0.20 mL: 0.025-0.05 mL: 50 mL; the stirring temperature is 30°C, the time is 30s, and the standing time is 12h. Deionized water A and deionized water B are both deionized water, and the different letters are only used to distinguish the names.

[0033] Preferably, in step 4 (a), the NH 4 The concentration of the UCNPs1 solution was 2 mg / mL; the NH 4-UCNPs1 solution, glutaraldehyde, PBS buffer B, avidin, PBS buffer C, PBS buffer D are used in a ratio of 5 mL: 1.25 mL: 5 mL: 0.5 mg: 5 mL: 5 mL, and the concentrations of PBS buffer A, PBS buffer B, PBS buffer C, and PBS buffer D are all 10 mmol / mL; the different letters in PBS buffer A, PBS buffer B, PBS buffer C, and PBS buffer D are only used to distinguish the names;

[0034] Preferably, in step 4 (a), the temperature of the shaker for reaction is 26°C, the reaction time is 2h, and the shaking incubation time is 12h; the amount of PBS buffer solution C added to RNA1 is 5mL:0.8-1.2mL, the concentration of RNA1 is 40-60nmol / L, and the temperature for overnight incubation is 37°C.

[0035] Preferably, in step 4 (a) and (b), the concentrations of NH4-UCNPs1-RNA1 and NH4-UCNPs1-RNA2 are both 1.8-2.1 mg / mL.

[0036] Preferably, in step 4 (c), the dosage relationship between the AuNRs solution and NH4-UCNPs1-RNA1 is 1mL:5.8-6.2mL; the dosage relationship between the AuNRs solution and NH4-UCNPs1-RNA2 is 1mL:5.8-6.2mL, the incubation temperature is 37°C, and the time is 12h; the dosage of the PBS buffer is 50mL; the concentrations of the Probe-RNA1 and Probe-RNA2 are both 1.6-2mg / mL.

[0037] Preferably, in step 4 (d), the dosage relationship of the DNA1-cDNA1, DNA2-cDNA2, OTA solution and AFB1 solution is 1.5mL:1.5mL:0.5mL:0.5mL, the concentrations of the DNA1-cDNA1 and DNA2-cDNA2 are both 0.5μmol / L, and the concentrations of OTA and AFB1 solution, if any, are both 0.1-100μg / L;

[0038] Preferably, in step 4 (d), the added OTA solution, Probe-RNA1, Probe-RNA2 and RNase-H solution are added in a ratio of 0.5 mL: 0.5 mL: 0.5 mL: 0.2 μL; the concentration of the RNase-H solution is 60 U / μL; and the co-incubation time is 10-70 min.

[0039] Preferably, in step (V), the concentration range of OTA and AFB1 set by the gradient is 0.1-100 μg / L; and the established prediction model is a SiPLS model.

[0040] Preferably, in step (six), the dosage ratio of the sample extract, Probe-RNA1, Probe-RNA2, and RNase-H is 0.5mL:0.5mL:0.5mL:0.2μL; the concentration of the RNase-H solution is 60U / μL; the concentrations of the Probe-RNA1 and Probe-RNA2 are both 1.6-2mg / mL, and the co-incubation time is 10-70min.

[0041] The reagents involved in the present invention are all purchased from Sangon Biotech (Shanghai) Co., Ltd., and the aptamer sequences are shown in Table 1:

[0042] Table 1: Aptamer sequences involved in the present invention

[0043]

[0044]

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention improves the water solubility of upconversion nanomaterials to assemble aptamers; obtains a specific absorption wavelength by adjusting the aspect ratio of the synthesized AuNRs; constructs upconversion nanomaterials and AuNRs fluorescent probes through RNA and DNA aptamers; and specifically cuts RNA in the DNA-RNA hybrid chain through RNase-H.

[0047] 2. The up-conversion fluorescence used in the detection method disclosed in the present invention can be distinguished from the down-conversion fluorescence that may exist in the object being measured, effectively avoiding the interference of background fluorescence and improving the sensitivity of the sensor; the synthesized AuNRs have an absorption peak of 860nm and can undergo fluorescence resonance energy transfer with the up-conversion material to achieve quenching of the fluorescence signal; RNase-H achieves the purpose of cyclic amplification of the detection system.

[0048] 3. By detecting OTA and AFB1 standards of different concentrations, collecting upconversion fluorescence spectra, and determining the quantitative prediction model, within the range of 0.1-100 μg / L, the calibration set and prediction set of the model have a high correlation coefficient, the root mean square error of the interactive verification is small, and the detection limit is low, which provides broad application prospects for the simultaneous detection of OTA and AFB1 in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the construction of OTA and AFB1 specific detection system.

[0050] Figure 2 Optimization diagram of the reaction time of Probe-RNA1 and Probe-RNA2 with RNase-H (a) and the amount of AuNRs used (b).

[0051] Figure 3 Figure 2 shows the fluorescence spectra (a) and quantitative prediction model (b) of OTA and AFB1 detection at different concentrations. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. The present invention includes but is not limited to the following embodiments, and any equivalent replacement or refusal to improve under the spirit and principle of the present invention will be deemed to be within the protection scope of the present invention.

[0053] Embodiment 1:

[0054] Step 1: Preparation of two upconversion nanomaterials:

[0055] UCNPs1: Yttrium oxide, ytterbium oxide and erbium oxide were dissolved in 5 mL trifluoroacetic acid solution at a ratio of 78 mol:20 mol:2 mol, and sodium fluoride was added to obtain a mixed solution A, wherein the concentration of sodium fluoride in the mixed solution was 10 mmol / L; the mixed solution A was transferred to a three-necked flask containing 20 mL oleylamine and 20 mL 1-octadecene mixed organic solution, nitrogen was continuously filled and magnetic stirring was performed, and the mixture was heated at 320°C for 1 h and then cooled to room temperature; the mixture was fully dissolved and washed with 100 mL of a mixed solution of cyclohexane and ethanol (wherein the volume ratio of cyclohexane to ethanol was 2:1); the precipitate obtained by centrifugation after washing was repeatedly washed with pure water, and the precipitate obtained by centrifugation after washing was freeze-dried to finally obtain an upconversion nanopowder, which was recorded as UCNPs1;

[0056] UCNPs2: Yttrium oxide, ytterbium oxide and thulium oxide were dissolved in 5 mL of trifluoroacetic acid solution in a ratio of 78 mol:20 mol:2 mol. The subsequent steps and parameter conditions were the same as the preparation method of UCNPs1, and finally upconversion nanopowder was obtained, which was recorded as UCNPs2.

[0057] Step 2: Modification of upconversion nanomaterials:

[0058] NH 4-UCNPs1: 20 mg UCNPs1 was accurately weighed and dissolved in 60 mL isopropanol, 20 mL distilled water and 2.5 mL ammonia water were added in sequence, the mixture of 20 mL isopropanol and 60 μL tetraethyl orthosilicate was added dropwise, and the reaction was continued for 3 h. Then, 30 mL isopropanol and 200 μL 3-aminopropyltriethoxysilane were added dropwise, and the stirring was stopped. The mixture was allowed to stand at room temperature for 2 h. Finally, two upconversion nanoparticles with amino groups on the surface were obtained after centrifugation and freeze-drying, which were recorded as NH 4 -UCNPs1.

[0059] NH 4 -UCNPs2: Replace UCNPs1 in the above steps with UCNPs2, and the final product is recorded as NH 4 -UCNPs2.

[0060] Step 3: Preparation of gold nanorods (AuNRs) with specific emission wavelength:

[0061] Preparation of seed solution: Take 5 mL of 0.5 mmol / L HAuCl 4 ·4H 2 O and 5 mL of 0.2 mol / L CTAB, then add 0.6 mL of freshly prepared 10 mmol / L NaBH 4 , stirred vigorously for 2 minutes, the color changed from yellow to yellow-brown, and aged at 30°C for 30 minutes.

[0062] Preparation of the growth solution (AuNRs solution): 1.8 g CTAB was weighed, 0.2468 g sodium oleate was added, dissolved in 50 mL deionized water at 50 °C, cooled to 30 °C, 4.8 mL 4 mmol / L silver nitrate was added, and the mixture was allowed to stand at 30 °C for 15 min. 50 mL 1 mmol / L HAuCl was added. 4 ·4H 2 O, slowly stir until colorless, add 0.1875mL concentrated HCl, slowly stir, then add 0.25mL0.064mol / L ascorbic acid, stir for 30s, finally, take 0.04mL seed solution, stir for 30s at 30℃, let stand for 12h, centrifuge, separate, and redissolve in 50mL deionized water to obtain AuNRs solution for use.

[0063] Step 4: Construction of specific detection system:

[0064] Accurately weigh 10 mg of NH 4-UCNPs1 was dissolved in 5 mL PBS buffer by ultrasonication, 1.25 mL glutaraldehyde was added, and the mixture was placed in a shaker at 26 °C for 2 h. After centrifugation, 0.5 mg avidin was added, and the mixture was incubated for 12 h. After freeze-drying, the mixture was redispersed in 5 mL PBS buffer, and 50 nmol / L of the ribonucleic acid chains (RNA1 and RNA2) corresponding to the analytes (OTA and AFB1) were added respectively. The mixture was incubated at 37 °C overnight, centrifuged, the supernatant was discarded, and the mixture was redispersed in 5 mL PBS buffer to obtain NH 4 -UCNPs1-RNA1 and NH 4 -UCNPs1-RNA2.

[0065] Pipette 1mL AuNRs and add 5mL NH 4 -UCNPs1-RNA1 and NH 4 -UCNPs1-RNA2, incubated in a shaker at 37°C for 12 h, washed with PBS buffer, centrifuged, discarded the supernatant, and redispersed in 5 mL PBS buffer to obtain two fluorescent detection probes (Probe-RNA1 and Probe-RNA2).

[0066] Add 0.5 mL of OTA and AFB1 (concentrations were 0.1 μg / L, 0.4 μg / L, 1 μg / L, 2 μg / L, 4 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L) to 0.5 mL of Probe-RNA1 and Probe-RNA2, respectively, and then add 0.2 μL (60 U / μL) of RNase-H. After reacting for 60 minutes, the test solution was obtained, thereby constructing a specific detection system for OTA and AFB1.

[0067] Step 5: OTA and AFB1 detection:

[0068] Firstly, the fluorescence spectral information of OTA and AFB1 test solutions with known concentrations (0.1μg / L, 0.4μg / L, 1μg / L, 2μg / L, 4μg / L, 10μg / L, 50μg / L, 100μg / L) was collected. 18 fluorescence spectra were collected at each corresponding concentration, and the fluorescence spectral information was randomly divided into a calibration set and a prediction set in a ratio of 2:1. The prediction model was established by combining the joint interval partial least squares (SiPLS).

[0069] Step 6: Detection of OTA and AFB1 in corn:

[0070] Referring to the methods of GB 5009.96-2016 and GB 5009.22-2016, the corn samples were processed to prepare sample extracts; then, 0.5 mL of the sample extract was added to 0.5 mL of Probe-RNA1 and Probe-RNA2, and then 0.2 μL (60 U / μL) of RNase-H was added, and the fluorescence spectrum was collected after the reaction for 60 min; the collected fluorescence spectrum information was then imported into the established SiPLS prediction model, and the specific concentration values ​​of OTA and AFB1 in corn were calculated by the SiPLS model, thereby realizing the simultaneous detection of OTA and AFB1 in corn. The specific results are shown in Table 1.

[0071] The comparison results between the fluorescence detection method provided by the present invention and the national standard method are shown in Table 1. According to the detection results in the table, it can be seen that the detection results of the fluorescence detection method provided by the present invention are basically consistent with the detection results of the national standard method, which proves that the detection method provided by the present invention has high accuracy.

[0072] Table 1 Comparison results of the sensor constructed by the present invention and the national standard method for detecting OTA and AFB1 in corn

[0073]

[0074]

[0075] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification, characterized in that: The following steps are involved: Step 1: Preparation of upconversion nanomaterials: (a) Dissolve yttrium oxide, ytterbium oxide and erbium oxide in trifluoroacetic acid respectively, then add sodium fluoride to obtain a mixed solution A, transfer the mixed solution A to a solution containing oleylamine and 1-octadecene; continue to fill with nitrogen and perform magnetic stirring, heat the reaction and then cool to room temperature; then add a mixed solution of cyclohexane and ethanol for centrifugal washing; the precipitate obtained by centrifugation is then washed with pure water by centrifugation, and finally freeze-dried to obtain an upconversion nanomaterial, which is recorded as UCNPs1; (b) The method and parameter conditions are the same as step (a), except that the yttrium oxide, ytterbium oxide and erbium oxide in step (a) are replaced by yttrium oxide, ytterbium oxide and thulium oxide; and finally, an upconversion nanomaterial is obtained, which is recorded as UCNPs2; Step 2: Modification of upconversion nanomaterials: (a) The UCNPs1 obtained in step 1 is dissolved in isopropanol A, and then distilled water and ammonia water are added in sequence. After sealing, the mixture is stirred, and then a mixture of isopropanol B and tetraethyl orthosilicate is added dropwise. Stirring is continued for a period of time, and then isopropanol C and 3-aminopropyltriethoxysilane are added dropwise. After the addition, stirring is stopped, and the mixture is allowed to stand at room temperature for aging. After aging, centrifugation is performed, and then freeze-drying is performed to obtain upconversion nanoparticles with amino groups modified on the surface, which are recorded as NH4-UCNPs1. (b) The operation and parameter conditions of step (b) are the same as those of step (a), except that UCNPs1 is replaced by UCNPs2; finally, upconversion nanoparticles with amino groups modified on the surface are obtained, which are denoted as NH4-UCNPs2; Step 3: Preparation of AuNRs with specific emission wavelength: (a) mixing a chloroauric acid solution and a hexadecyltrimethylammonium bromide solution, adding a sodium borohydride solution, stirring for a period of time, and then standing at a certain temperature to obtain a seed solution; (b) dissolving hexadecyltrimethylammonium bromide and sodium oleate in deionized water A, then adding silver nitrate, and allowing to react to obtain a mixed solution A; adding chloroauric acid solution to the mixed solution A and stirring until colorless, then adding concentrated hydrochloric acid and ascorbic acid solution in sequence, stirring to obtain a mixed solution B; adding the seed solution prepared in step (a) to the mixed solution B, stirring, allowing to stand, centrifuging, and re-dissolving the obtained precipitated product in deionized water B to obtain an AuNRs solution with a specific emission wavelength; Step 4: Construction of specific detection system: (a) The NH4-UCNPs1 obtained in step 2 is added to PBS buffer A for ultrasonic dissolution to obtain NH4-UCNPs1 solution, glutaraldehyde is added, and the mixture is placed on a shaker for reaction. The precipitate after centrifugation is re-dispersed in PBS buffer B by ultrasonication, and then avidin is added and incubated by shaking. After incubation, the freeze-dried product is re-dispersed in PBS buffer C to obtain a mixed solution A, and finally the ribonucleic acid chain corresponding to the analyte OTA is added, which is recorded as RNA1, and the mixture is incubated overnight. The precipitate obtained after centrifugation is re-dispersed in PBS buffer D, and the obtained mixed solution is recorded as NH4-UCNPs1-RNA1; (b) The operation and parameter conditions are the same as those in step (a), except that NH4-UCNPs1 is replaced by NH4-UCNPs2, and the RNA chain RNA1 is replaced by the RNA chain corresponding to AFB1, i.e., RNA2, to finally obtain the product NH4-UCNPs2-RNA2; (c) The AuNRs solution prepared in step 3 was added to NH4-UCNPs1-RNA1 and NH4-UCNPs1-RNA2, respectively, to obtain mixed solution A and mixed solution B, which were placed in a shaker for incubation, washed with PBS buffer after incubation, and centrifuged after washing. The obtained products were recorded as product A and product B, respectively; product A and product B were then dispersed in PBS buffer solution to obtain two fluorescent detection probe solutions, which were recorded as Probe-RNA1 and Probe-RNA2, respectively; (d) Select two base-complementary paired DNA1-cDNA1 and DNA2-cDNA2 and mix them with a certain concentration of OTA solution and AFB1 solution; then add Probe-RNA1 and Probe-RNA2 obtained in step (c); finally add RNase-H for co-incubation, and obtain the test solution after incubation, that is, construct the OTA and AFB1 specific detection system; Step 5: OTA and AFB1 detection: Take the test solution prepared in step 4, set the concentration of OTA and AFB1 therein in a gradient, collect fluorescence spectrum information of the test solution with known OTA and AFB1 concentrations, and establish a prediction model based on the fluorescence spectrum information and concentration; Step 6, detection of OTA and AFB1 in the sample: Referring to the methods of GB 5009.96-2016 and GB 5009.22-2016, the sample was processed to prepare a sample extract; then, the sample extract was added to Probe-RNA1 and Probe-RNA2, and then RNase-H was added for co-incubation to obtain a sample test solution after incubation, and the fluorescence spectrum of the sample test solution was collected; The collected fluorescence spectrum information is then imported into the prediction model established in step five; the specific concentration values ​​of OTA and AFB1 in the sample are obtained through model calculation, thereby achieving simultaneous detection of OTA and AFB1 in the sample.

2. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 1 (a), the dosage ratio of yttrium oxide, ytterbium oxide, erbium oxide and trifluoroacetic acid is 74-80 mol:18-24 mol:1.8-2.2 mol:5 mL; the concentration of sodium fluoride in the mixed solution A is 10 mmol / L; the dosage ratio of trifluoroacetic acid to oleylamine and 1-octadecene in the mixed solution A is 5 mL:20 mL:20 mL; the dosage ratio of the mixed solution of cyclohexane and ethanol to trifluoroacetic acid in the mixed solution A is 100-200 mL:5 mL; the volume ratio of cyclohexane and ethanol is 2:1; the temperature of the heating reaction is 320°C, and the reaction time is 1 h.

3. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 2 (a), the dosage ratio of UCNPs1, isopropanol A, distilled water, and ammonia water is 20 mg: 50-65 mL: 15-25 mL: 2-3 mL; the dosage ratio of ammonia water to isopropanol B, tetraethyl orthosilicate, isopropanol C, and 3-aminopropyltriethoxysilane is 2-3 mL: 17-22 mL: 50-70 μL: 25-33 mL: 180-200 μL; the continuous stirring period is 3 h; and the aging time at room temperature is 2 h.

4. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step three (a), the dosage ratio of the chloroauric acid solution, the hexadecyltrimethylammonium bromide solution and the sodium borohydride solution is 5 mL:5 mL:0.6 mL; the concentration of the chloroauric acid solution is 0.5 mmol / L, the concentration of the hexadecyltrimethylammonium bromide solution is 0.2 mol / L, and the concentration of the sodium borohydride solution is 10 mmol / L; the stirring period is 2 to 5 min; the temperature of standing under the certain temperature condition is 30°C and the time is 30 min.

5. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 3 (b), the ratio of hexadecyltrimethylammonium bromide, sodium oleate, deionized water A and silver nitrate is 1.8 g:0.2468 g:50 mL:4.8 mL; the concentration of silver nitrate is 4 mmol / L; the conditions for adding silver nitrate and standing the reaction are: temperature 30°C, reaction time 15 min; The ratio of deionized water A to HAuCl4·4H2O, concentrated hydrochloric acid, and ascorbic acid solution in the mixed solution A is 50 mL:50 mL:0.15-0.20 mL:0.15-0.20 mL; the concentration of HAuCl4·4H2O is 1 nmol / L, and the concentration of ascorbic acid solution is 0.064 mol / L; the stirring time after adding concentrated hydrochloric acid and ascorbic acid is 30-40 s; The dosage ratio of ascorbic acid solution, seed solution and deionized water B in the mixed solution B is 0.15-0.20 mL:0.025-0.05 mL:50 mL; the stirring temperature is 30° C., the stirring time is 30 s, and the standing time is 12 h.

6. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 4 (a), the concentration of the NH4-UCNPs1 solution is 2 mg / mL; the dosage ratio of the NH4-UCNPs1 solution, glutaraldehyde, PBS buffer B, avidin, PBS buffer C, and PBS buffer D in the mixed solution A is 5 mL:1.25 mL:5 mL:0.5 mg:5 mL:5 mL, and the concentrations of the PBS buffer A, PBS buffer B, PBS buffer C, and PBS buffer D are all 10 mmol / mL; The temperature of the shaker for the reaction is 26°C, the reaction time is 2 h, and the shaking incubation time is 12 h; the amount of the added PBS buffer solution C and RNA1 is 5 mL:0.8-1.2 mL, the concentration of the RNA1 is 40-60 nmol / L, and the temperature for overnight incubation is 37°C; In step 4 (a) and (b), the concentrations of NH4-UCNPs1-RNA1 and NH4-UCNPs1-RNA2 are both 1.8-2.1 mg / mL.

7. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 4 (c), the dosage relationship of the AuNRs solution and NH4-UCNPs1-RNA1 is 1 mL:5.8-6.2 mL; the dosage relationship of the AuNRs solution and NH4-UCNPs1-RNA2 is 1 mL:5.8-6.2 mL, the incubation temperature is 37°C, and the time is 12 h; the dosage of the PBS buffer is 50 mL; the concentrations of the Probe-RNA1 and Probe-RNA2 are both 1.6-2 mg / mL.

8. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step 4 (d), the dosage ratio of the DNA1-cDNA1, DNA2-cDNA2, OTA solution and AFB1 solution is 1.5 mL:1.5 mL:0.5 mL:0.5 mL, the concentrations of the DNA1-cDNA1 and DNA2-cDNA2 are both 0.5 μmol / L, and the concentrations of OTA and AFB1 solution, if any, are both 0.1-100 μg / L; the dosage ratio of the added OTA solution, Probe-RNA1, Probe-RNA2 and Rnase-H solution is 0.5 mL:0.5 mL:0.5 mL:0.2 μL; the concentration of the Rnase-H solution is 60 U / μL; and the co-incubation time is 10-70 min.

9. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step (V), the concentration range of OTA and AFB1 in the gradient setting is 0.1-100 μg / L; the established prediction model is the SiPLS model.

10. The method for simultaneous detection of aflatoxins and ochratoxins based on upconversion fluorescence cyclic amplification according to claim 1, characterized in that: In step (six), the dosage ratio of the sample extract, Probe-RNA1, Probe-RNA2, and RNase-H is 0.5 mL: 0.5 mL: 0.5 mL: 0.2 μL; the concentration of the RNase-H solution is 60 U / μL; the concentrations of the Probe-RNA1 and Probe-RNA2 are both 1.6-2 mg / mL, and the co-incubation time is 10-70 min.

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