A ratiometric fluorescence biosensor based on gold nanoclusters and its preparation method and application in detecting Aspergillus fungi

Abstract

The present invention relates to a ratio fluorescence biosensor based on gold nanoclusters, a preparation method thereof and an application thereof in detecting Aspergillus fungi. The present invention takes the DNA genome of Aspergillus fungi as a target, designs LAMP primers, and adds FITC@AuNCs to perform LAMP reaction on the basis of determining the primer specificity. AuNCs protected by BSA are used as fluorescent groups, and FITC is used as a pH response signal. As the LAMP reaction proceeds, a large amount of pyrophosphate and hydrogen ions are released, resulting in a gradual decrease in the ratio fluorescence signal of FITC and AuNCs, which can be used for quantitative analysis of Aspergillus fungi detection.

Description

Technical Field

[0001] The present invention relates to the field of food safety biotechnology, and in particular to a ratiometric fluorescent biosensor based on gold nanoclusters, a preparation method thereof, and an application thereof in detecting Aspergillus molds. Background Art

[0002] Snack foods are foods that people eat during breaks and rest, and are mainly divided into cereal products, dried fruit products, confectionery products, and meat products. In 2020, the total output value of the snack food industry reached 3 trillion yuan, and it is expected to exceed 4 trillion yuan in 2025. It can be seen that the snack food industry is developing rapidly and has great development potential. Among them, the annual output value of the nuts and roasted seeds industry has rapidly increased from 45.2 billion yuan to 121.4 billion yuan, with an average annual compound growth rate of 15.2%, which is much higher than the overall snack food. At the same time, since 2017, 198 cases of excessive mold in food have been investigated and reported across the country. The cases of excessive mold in nuts and roasted seeds accounted for 20% of the total number of reports, and this problem has gradually become one of the bottlenecks in the development of the industry. Aspergillus, as a common saprophytic fungus, can not only produce a large number of spores, but also has different biochemical activities. Therefore, it is widely distributed and can grow in different environments and substrates, especially in plant products in humid and warm environments to reproduce and produce mycotoxins. However, Aspergillus flavus has been reported in mold contamination of cashews, pistachios, chestnuts and almonds, and is the main pollutant causing mold on nuts.

[0003] At present, the commonly used methods for detecting mold are traditional culture method and biochemical detection technology. The traditional culture method is the national standard GB4789.15-2016 "National Food Safety Standard Food Microbiology Test Mold and Yeast Count". The traditional culture method has accurate results, but the detection cycle is long and the operation steps are cumbersome, which is not suitable for the needs of rapid and efficient detection. Biochemical detection technology is to detect the type and quantity through the specific reaction between the metabolites produced by the growth and reproduction of mold and specific substrates. Commonly used detection methods include rapid test strip method and ATP bioluminescence technology detection method. Although this type of method is simple to operate and low cost, the accuracy of the detection results is poor.

[0004] Molecular biology technology provides new ideas and important basis for the detection of food mold based on the relationship and interaction between nucleic acids and their expression products. At present, the PCR-based detection method is still the preferred method for detecting mold in food. The effectiveness of the PCR-based detection method depends on its specific in vitro amplification of the target DNA sequence, which has high specificity and sensitivity. However, PCR detection cannot achieve quantitative results and can only remain at the qualitative stage. At the same time, aerosol contamination caused by opening the lid during the detection process will cause false positive test results. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a ratio fluorescence biosensor based on gold nanoclusters, a preparation method thereof, and its application in detecting Aspergillus fungi. The present invention takes the Aspergillus fungi genome as a target, aligns the sequence by DNAMAN software, designs LAMP primers by software such as NEB LAMP Primer Design Tool, and adds FITC@AuNCs to perform LAMP reaction on the basis of determining the primer specificity. Using BSA-protected AuNCs as a fluorescent group and FITC as a pH response signal, as the LAMP reaction proceeds, a large amount of pyrophosphate and hydrogen ions are released, resulting in a gradual decrease in the ratio fluorescence signal of FITC and AuNCs, which can be used for quantitative analysis of nucleic acid detection.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a ratiometric fluorescent biosensor based on gold nanoclusters, the ratiometric fluorescent biosensor comprising a primer probe and FITC@AuNCs, the primer probe comprising an upstream outer primer, a downstream outer primer, an upstream inner primer, a downstream inner primer, an upstream loop primer and a downstream loop primer;

[0008] The nucleotide sequence of the upstream outer primer is shown in SEQ ID No.1, the nucleotide sequence of the downstream outer primer is shown in SEQ ID No.2, the nucleotide sequence of the upstream inner primer is shown in SEQ ID No.3, the nucleotide sequence of the downstream inner primer is shown in SEQ ID No.4, the nucleotide sequence of the upstream loop primer is shown in SEQ ID No.5, and the nucleotide sequence of the downstream loop primer is shown in SEQ ID No.6.

[0009] In one embodiment of the present invention, the FITC@AuNCs comprises a gold nanocluster material cross-linked by FITC, wherein the diameter of the gold nanocluster cross-linked by FITC is about 5-8 nm.

[0010] In one embodiment of the present invention, the FITC@AuNCs are prepared by the following method:

[0011] HAuCl4 and BSA solutions are mixed, and the pH of the reaction solution is adjusted to alkaline, and the reaction is stirred to obtain AuNCs; the obtained AuNCs are dissolved in a buffer solution to obtain an AuNCs solution; an alcohol solution of FITC is added to the AuNCs solution, and the mixture is stirred to obtain the FITC@AuNCs.

[0012] In one embodiment of the present invention, the pH value is 9-11.

[0013] In one embodiment of the present invention, the concentration of the FITC alcohol solution is 1-2 mg / mL.

[0014] The second object of the present invention is to provide a kit comprising the ratiometric fluorescence biosensor.

[0015] The third object of the present invention is to provide the use of the ratio fluorescence biosensor and the kit in the quantitative or qualitative detection of mold.

[0016] The detection limit of the kit for Aspergillus is 7.4×10 -3 ng / mL, the detection linear range is 10 -2 ng / mL-10 2 ng / mL.

[0017] Furthermore, a method for detecting Aspergillus fungi in nuts using the kit comprises the following steps:

[0018] (1) Verification of specificity of designed primers

[0019] The genomes of standard samples of Aspergillus, Penicillium and Fusarium were extracted respectively, primer combinations were added, 25μL LAMP system was configured, and the LAMP program was set to perform amplification experiments. The specificity of the designed primers was determined by detecting the electrophoresis results of the amplified products of the fungi contained in the samples.

[0020] (2) Establishment of the Aspergillus standard curve

[0021] Prepare different concentrations of Aspergillus standard genomes, add primer combinations, prepare 25μL LAMP system, set up LAMP program to perform amplification experiments, and establish standard curves corresponding to Aspergillus fungi by detecting the fluorescence response of standard solutions of different concentrations after the reaction.

[0022] (3) Detection of Aspergillus fungi in nuts:

[0023] After pretreatment, the nut samples were cultured in liquid culture medium, the mycelium in the culture medium was broken by liquid nitrogen, the sample genome was extracted from the broken tissue using the kit method, a primer probe combination was added, a 25 μL LAMP system was configured, and the LAMP program was set to perform an amplification experiment. The fluorescence response of the nut samples after the reaction was detected and combined with the corresponding standard curve to calculate the concentration of the Aspergillus fungus genome contained in the nuts.

[0024] In one embodiment of the present invention, the mold is selected from Aspergillus, Penicillium or Fusarium.

[0025] In one embodiment of the present invention, the Aspergillus fungus is Aspergillus niger, Aspergillus flavus, Aspergillus jifengensis, the Penicillium fungus is Penicillium viridans, and the Fusarium fungus is Fusarium oxysporum.

[0026] In one embodiment of the present invention, the application method comprises the following steps: mixing the primer probe, the FITC@AuNCs and the DNA template of Aspergillus fungus to perform LAMP reaction, taking the LAMP product after the reaction is completed, and detecting the fluorescence intensity of the product.

[0027] In one embodiment of the present invention, the LAMP reaction further comprises isothermal amplification buffer, dNTPs, DNA polymerase and water.

[0028] In one embodiment of the present invention, the ratiometric fluorescence biosensor is used to detect Aspergillus fungi in nuts.

[0029] In one embodiment of the present invention, the method for detecting Aspergillus in nuts comprises the following steps:

[0030] S1: Add nuts containing Aspergillus fungi into sterilized water and shake them, add the shaken solution into culture medium for cultivation to obtain hyphae, and extract sample genomic DNA through the hyphae;

[0031] S2: Perform LAMP reaction on the sample genomic DNA prepared in S1, the primer probe and the FITC@AuNCs solution, and after the reaction is completed, take the LAMP product for fluorescence detection.

[0032] The present invention is different from the temperature change requirements brought about by denaturation, annealing and extension of PCR. The loop-mediated isothermal amplification technology is a highly sensitive constant temperature nucleic acid amplification technology. In the early stage, a pair of outer primers and a pair of inner primers are designed for the six regions of the target DNA chain. A pair of loop primers can also be designed between the inner primer design regions to improve the detection efficiency. Add the target nucleic acid, primers, Bst DNA polymerase and dNTPs to the system, keep the whole system at about 63°C for about 1 hour, and complete the nucleic acid amplification process through chain displacement reaction.

[0033] Mechanism of the present invention:

[0034] The present invention uses BSA-protected AuNCs as a reference signal and conjugates with FITC to form a ratiometric fluorescent probe FITC@AuNCs for pH detection. Here, FITC is used as a specific recognition element for pH, and its fluorescence emission center is 517nm. When pH increases (OH - When the pH value is lowered (H+ When the concentration increases, the lactone ring will close and be accompanied by weak fluorescence, while the red fluorescence at 625 nm attributed to AuNCs remains unchanged. Therefore, considering the pH change caused by LAMP during the amplification process, the AuNCs ratio fluorescence method was combined with LAMP to determine the quantitative detection of this method during the LAMP reaction.

[0035] The technical solution of the present invention has the following advantages:

[0036] (1) The present invention compares gene sequences of different fungi in the gene sequence library of NCBI and designs universal LAMP primers for Aspergillus at conserved sites to ensure that it has the versatility to detect common Aspergillus fungi in nuts.

[0037] (2) The present invention performs quantitative detection based on the change in the ratio fluorescence value of FITC@AuNCs, which can eliminate the influence of the difference in local probe concentration and distribution changes, thereby improving the accuracy of detection. The ratio fluorescence value refers to the ratio of the fluorescence emission value of FITC at 625nm to the fluorescence emission value of AuNCs at 517nm under 488nm ultraviolet excitation light.

[0038] (3) The present invention extracts DNA of Aspergillus fungi in nuts and performs LAMP amplification to achieve qualitative and quantitative analysis. The operation is simple and rapid, and the entire detection process can be completed within a few hours.

[0039] (4) The detection conditions of the present invention are mild, avoiding the large instruments required for heating and cooling in the ordinary PCR detection process, making the operation more portable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0041] Figure 1 The characterization diagram of AuNCs and FITC@AuNCs in Example 1 of the present invention; wherein Figure 1 (a) and 1(b) are TEM images of AuNCs and FITC@AuNCs, respectively; Figure 1 (c) XRD patterns of AuNCs and FITC@AuNCs; Figure 1 (d) FT-IR images of AuNCs and FITC@AuNCs; Figure 1 (e) UV-visible absorption spectra of AuNCs and FITC@AuNCs; Figure 1 (f) Fluorescence spectra of AuNCs and FITC@AuNCs;

[0042] Figure 2The 28S rRNA gene sequence Blast comparison result in Example 2 of the present invention;

[0043] Figure 3 The 28S rRNA gene sequence and the position and orientation of the primers thereof in Example 2 of the present invention;

[0044] Figure 4 The specificity verification and sensitivity detection of LAMP primers in Example 2 of the present invention;

[0045] Figure 5 5(a) is a characterization diagram of LAMP in Example 3 of the present invention; wherein 5(a) is a characterization diagram of LAMP reaction temperature optimization: M: 10 kbp DNA Marker; lanes 1-6: 55°C, 57°C, 59°C, 61°C, 63°C, 65°C; N: negative control; 5(b) is a characterization diagram of LAMP reaction time optimization: M: 10 kbp DNA Marker; lanes 1-6: 45 min, 50 min, 55 min, 60 min, 65 min, 70 min; N: negative control;

[0046] Figure 6 This is to verify the feasibility of the LAMP primers in Example 3 of the present invention;

[0047] Figure 7 This is the sensitivity detection and standard curve of FITC@AuNCs-LAMP in Example 3 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] In the following examples, the primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.:

[0050] The nucleotide sequence of the upstream outer primer is (as shown in SEQ ID No.1):

[0051] 5'-GCAGAGGGTACTTTGGGT-3'

[0052] The nucleotide sequence of the downstream outer primer is (as shown in SEQ ID No.2):

[0053] 5'-CTCTACTTGTGGCGTATCGG-3'

[0054] The nucleotide sequence of the upstream inner primer is (as shown in SEQ ID No.3):

[0055] 5'-CTTTACACGGGCACGGACACCTAATCGTGCCCTGGAACGG-3'

[0056] The nucleotide sequence of the downstream inner primer is (as shown in SEQ ID No.4):

[0057] 5'-TCGACGAGTCGAGTTGGGGTTTTCTCCGGCCAGTATTTAGC-3'

[0058] The nucleotide sequence of the upstream loop primer is (as shown in SEQ ID No.5):

[0059] 5'-CCAAGACGTAGGTCTCACC-3'

[0060] The nucleotide sequence of the downstream loop primer is (as shown in SEQ ID No.6):

[0061] 5'-AATGCAGCAAATCTTGGGTGG-3'

[0062] The Aspergillus, Penicillium and Fusarium used in the present invention are specifically Aspergillus niger CICC 2487, Aspergillus flavus CICC 2219, Penicillium viridicatum CICC 4029, and Fusarium oxysporum CICC 41029, which are all purchased from the China Industrial Microbiological Culture Collection Center, and Aspergillus nomius ACCC 32558 is purchased from the China Agricultural Microbiological Culture Collection Center.

[0063] Example 1 Synthesis and characterization of FITC@AuNCs

[0064] Gold nanoclusters (AuNCs): 10 mL of HAuCl4 (10 mmol / L) was added to 10 mL of BSA solution (50 mg mL -1 ), and then 1 mL of NaOH solution (1 mol / L) was added. After the reaction was continued for 12 h under vigorous stirring, the AuNCs were dialyzed in ultrapure water for 24 h (the water was changed every 8 h), and the prepared AuNCs were stored at 4 °C for use. For material characterization and subsequent synthesis, the solid AuNCs were dried using a freeze dryer and redispersed in PBS buffer (pH = 7.4) and stored at 4 °C for use.

[0065] FITC@AuNCs: 1 mg / mL FITC ethanol solution was added to AuNCs reconstituted with PBS buffer (pH = 7.4), stirred in the dark at room temperature for 12 h, and then dialyzed with 0.05 mol / L PBS buffer (pH = 7.4) for 8 h to obtain the product FITC@AuNCs, which was stored in a 4 °C refrigerator.

[0066] The successful connection between FITC and AuNCs was verified by FT-IR and UV-Vis spectroscopy. Figure 1 (a) and (b) are TEM images of AuNCs and FITC@AuNCs, respectively. Figure 1 (a) and 1(b) show that the prepared AuNCs are a monodisperse system with an average particle size of about 5 nm. At the same time, after combining with FITC, its dispersibility has basically not changed.

[0067] Figure 1 (c) is the XRD pattern of AuNCs and FITC@AuNCs. Figure 1 (c) It can be seen that the characteristic diffraction peak (111) of AuNCs at 38.69° corresponds well to the standard cubic phase of Au (standard card PDF#04-0784). When combined with FITC, the diffraction peak shifts to 32.19°, which may be related to the increase in the lattice of Au.

[0068] Figure 1 (d) is the FT-IR image of AuNCs and FITC@AuNCs. Figure 1 (d) It can be seen that after FITC is conjugated to the surface of AuNCs, the peak appears at 1662 cm -1 (V C＝O ) and 2042cm -1 (V N＝C＝S ). The infrared spectrum shows that FITC is successfully covalently bound to BSA-AuNCs.

[0069] Figure 1 (e) is the UV-visible absorption spectra of AuNCs and FITC@AuNCs. Figure 1 (e) It can be seen that BSA has an ultraviolet absorption peak at 280nm. When it synthesizes BSA-AuNCs, the absorption peak at 280nm decreases. In addition, the absorption peak of FITC@AuNCs at around 280nm also indicates the binding with BSA.

[0070] Figure 1 (f) is the fluorescence spectra of AuNCs and FITC@AuNCs. Figure 1(f) It can be seen that under the excitation of 488 nm excitation light, the fluorescence emission of AuNCs was observed to be concentrated at 640 nm, and the color of the solution was bright red (see Figure 1 (a) Illustration). When combined with FITC, the color of the solution changes from orange-yellow to yellow-green. Under the same wavelength of excitation light, its excitation emission peak shifts to 630nm, and the color of the solution becomes orange (see Figure 1 (b) Inset). Meanwhile, the emission peak of FITC shifted from 520 nm to 521 nm, indicating the successful conjugation of FITC and AuNCs.

[0071] Example 2 Design and verification of primers

[0072] (1) Extract genomic DNA of the test strain using the kit method

[0073] Activated Aspergillus niger, Aspergillus jifengensis, Penicillium viridum and Fusarium oxysporum were cultured in potato liquid culture medium at 25°C and 180r / min shaking for 40h, and Aspergillus flavus was cultured in malt juice liquid culture medium at 28°C and 180r / min shaking for 40h. The mycelial pellets in the liquid culture medium were extracted with a kit (the kit here is Biospin Fungal Genome Extraction Kit purchased from Hangzhou Biori Technology Co., Ltd.). The specific operation is as follows:

[0074] a: The mortar, pestle and medicine spoon are sterilized in advance, and the test bench is wiped with 75% alcohol. Pour some liquid nitrogen into the mortar to pre-cool the mortar and pestle. Then take out the aflatoxin mycelium balls suspended in the liquid culture medium and pour them into the mortar, crush the fungal tissue into powder, and continuously add liquid nitrogen during the process to prevent the liquid nitrogen from completely vaporizing;

[0075] b: Add a small amount of ground fungal tissue to a 1.5 mL centrifuge tube; add 400 μL LEBuffer and 4 μL 20 mg / mL RNase A in an ice bath and mix well;

[0076] c: Place the centrifuge tube in a 65°C water bath for 30 minutes, vortexing the centrifuge tube every 10 minutes to ensure uniform oscillation of the solution;

[0077] d: Add 130 μL DABuffer to the centrifuge tube, vortex to mix evenly, place in an ice bath for 5 min, centrifuge at 15,000 rpm for 3 min, and use a pipette to draw the supernatant and add it to a new 1.5 mL centrifuge tube;

[0078] e: Add 750 μL of E Binding Buffer to the centrifuge tube, vortex to mix evenly, transfer the mixed liquid to the nucleic acid purification column twice, centrifuge at 10400 r / min for 1 min, and discard the liquid in the liquid collection tube;

[0079] f: Add 500 μL of G Binding Buffer, 600 μL of Wash Buffer, and 600 μL of Wash Buffer to the nucleic acid purification column in sequence. Centrifuge at 13400 rpm for 30 seconds after each addition, and discard the liquid in the liquid receiving tube.

[0080] g: Centrifuge the nucleic acid purification column at 13400r / min for 1min, discard the liquid collection tube, and transfer the nucleic acid purification column to a new 1.5mL centrifuge tube;

[0081] h: Add about 150 μL of Elution Buffer to the nucleic acid purification column and incubate at about 23°C for 1 minute;

[0082] i: After the nucleic acid purification column was centrifuged at 14700r / min for 1 min, the centrifuge tube was retained. The centrifuge tube contained the extracted genomic DNA of Aspergillus flavus and was stored at -20°C. Similarly, the genomic DNA of Aspergillus niger, Aspergillus jifengensis, Penicillium virens and Fusarium oxysporum were obtained and stored at -20°C. The extracted whole genome was used as a template and the concentration and purity of the extracted DNA were determined by Nanodrop.

[0083] (2) Design of primers and probes

[0084] The gene sequences of Aspergillus flavus, Aspergillus niger, Aspergillus nomius and other molds commonly found in nuts were downloaded from the NCBI database. The 28S rRNA sequences of six Aspergillus molds (GenBank: NG_069611.1, NG_055742, NG_055744.1, D84354.2, AB008418.1 and NG_069608.1) were compared using DNAMAN V6 software. Blast analysis was performed on the highly similar parts to confirm that they had the specificity for Aspergillus detection. The analysis results are shown in Figure 2. Figure 2 As shown in Figure 2, the 28S rRNA sequence has good specificity for Aspergillus detection and can be used for LAMP primer design. The screened gene fragments were imported into NEB LAMP Primer Design in FASTA format to automatically generate three sets of LAMP primer pairs. The primer specificity was analyzed by Blast and the mismatch between primers was verified by NUPACK. Finally, the primer pairs for the invention (F3, B3, FIP and BIP) were determined. The primer sequences were then imported into Primer Explorer V5 to determine two pairs of loop primers (LF and LB). The positions of each primer in the target sequence are shown in Figure 2. Figure 3 shown.

[0085] F3: The nucleotide sequence of the upstream outer primer is (as shown in SEQ ID No.1):

[0086] 5'-GCAGAGGGTACTTTGGGT-3'

[0087] B3: The nucleotide sequence of the downstream outer primer (SEQ ID No. 2) is:

[0088] 5'-CTCTACTTGTGGCGTATCGG-3'

[0089] FIP: The nucleotide sequence of the upstream inner primer (SEQ ID No. 3) is:

[0090] 5'-CTTTACACGGGCACGGACACCTAATCGTGCCCTGGAACGG-3'

[0091] BIP: The nucleotide sequence of the downstream inner primer (SEQ ID No. 4) is:

[0092] 5'-TCGACGAGTCGAGTTGGGGTTTTCTCCGGCCAGTATTTAGC-3'.

[0093] LF: The nucleotide sequence of the upstream loop primer is (as shown in SEQ ID No.5):

[0094] 5'-CCAAGACGTAGGTCTCACC-3'

[0095] LB: The nucleotide sequence of the downstream loop primer is (as shown in SEQ ID No.6):

[0096] 5'-AATGCAGCAAATCTTGGGTGG-3'

[0097] (3) Primer specificity verification

[0098] The LAMP reaction system was 25 μL, including 8 μL 2×isothermal amplification buffer, 1.25 μL dNTPs, 1 μL Bst 2.0 DNA polymerase (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), 0.6 μL each of the outer primer and inner primer, 0.3 μL each of the loop primer, 1.25 μL of the DNA templates of Aspergillus, Penicillium and Fusarium extracted in step (1) of Example 2, 3.5 μL FITC@AuNCs, and 3.5 μL sterile ultrapure water. The amplification procedure was: constant temperature at 63°C for 55 min; enzyme inactivation at 80°C for 5 min. The 2.5 μL LAMP product was electrophoresed on a 1% agarose gel, stained with GelRed, and the result was determined according to the band size under ultraviolet light. If a ladder-type length product can be specifically amplified, it can be determined that the designed primers can be used for the detection of the corresponding bacterial genus.

[0099] The LAMP test was performed on 5 species of Aspergillus, Penicillium and Fusarium. The test results were as follows: Figure 4 As shown, lanes 1'-3' where the target genes extracted from the three Aspergillus species all showed continuous LAMP ladder bands, while lanes 4' and 5' where the DNA of Penicillium and Fusarium were located did not show any bands. This verifies that the designed LAMP primers can be used for subsequent quantitative detection.

[0100] The nucleotide sequences of the primers designed by the present invention are as follows:

[0101] The nucleotide sequence of the upstream outer primer (SEQ ID No.1) is:

[0102] 5'-GCAGAGGGTACTTTGGGT-3'

[0103] The nucleotide sequence of the downstream outer primer (SEQ ID No.2) is:

[0104] 5'-CTCTACTTGTGGCGTATCGG-3'

[0105] The nucleotide sequence of the upstream inner primer (SEQ ID No.3) is:

[0106] 5'-CTTTACACGGGCACGGACACCTAATCGTGCCCTGGAACGG-3'

[0107] The nucleotide sequence of the downstream inner primer (SEQ ID No.4) is:

[0108] 5'-TCGACGAGTCGAGTTGGGGTTTTCTCCGGCCAGTATTTAGC-3'

[0109] The nucleotide sequence of the upstream loop primer (SEQ ID No.5) is:

[0110] 5'-CCAAGACGTAGGTCTCACC-3'

[0111] The nucleotide sequence of the downstream loop primer (SEQ ID No.6) is:

[0112] 5'-AATGCAGCAAATCTTGGGTGG-3'.

[0113] Example 3 FITC@AuNCs-LAMP sensitivity detection and establishment of standard curve

[0114] (1) Extracting genome

[0115] According to the method described in step (1) of Example 2, the genomes of Aspergillus niger, Aspergillus flavus, Aspergillus jifengensis, Penicillium viride and Fusarium oxysporum were extracted respectively. The concentrations of the obtained Aspergillus niger genome were 7.2 ng / μL, the concentration of Aspergillus flavus was 7.2 ng / μL, the concentration of Aspergillus jifengensis genome was 6.9 ng / μL, the concentration of Penicillium viride genome was 3.3 ng / μL, and the concentration of Fusarium oxysporum genome was 14 ng / μL.

[0116] (2) Optimization of FITC@AuNCs-LAMP reaction conditions

[0117] The amplification conditions of FITC@AuNCs-LAMP reaction were explored, and electrophoresis was performed to verify the reaction temperature of 55℃-65℃ and the reaction time of 45min-70min. Figure 5 As shown, bands appeared at reaction temperatures of 55°C-65°C. At the same time, the brightness of each band was similar within the time range of 45min-70min. The system was subsequently verified for repeatability, and the optimized temperature was determined to be 63°C and the optimized reaction time to be 55min.

[0118] (3) Feasibility verification of FITC@AuNCs-LAMP reaction

[0119] Specific operation: For the obtained LAMP product, the fluorescence value was analyzed by F97 pro fluorescence analyzer. The fluorescence analyzer conditions were set as: excitation light 488nm, emission light range 500nm-700nm. The ratio fluorescence value refers to the ratio of the fluorescence emission value of FITC at 625nm to the fluorescence emission value of AuNCs at 517nm under 488nm ultraviolet excitation light. The results are shown in Figure 6 .

[0120] Depend on Figure 6It can be seen that the ratio fluorescence values ​​of nucleic acid of standard strains of Aspergillus niger, Aspergillus flavus and Aspergillus jifengensis were around 4.8, while the ratio fluorescence values ​​of negative control Penicillium viridans and Fusarium oxysporum were maintained above 5.8. Figure 6 (a) It can be seen that the green emission peak intensity of Aspergillus fungi at 517 nm is less than that of the blank control and the negative control, indicating that this method can be used to detect specific recognition in LAMP reactions.

[0121] (4) FITC@AuNCs-LAMP sensitivity detection and establishment of standard curve

[0122] The concentration of Aspergillus niger DNA at 7.4 ng / μL was diluted 10 times, and 7 concentration gradients were set to carry out FITC@AuNCs-LAMP experiment. The ratio fluorescence values ​​under 7 concentration gradients were measured respectively, and the standard curve was drawn according to the size of the ratio fluorescence value (FGreen / FRed). Three parallel experiments were carried out for each concentration.

[0123] The experimental results are as follows Figure 7 As shown in the figure, in this ratio biosensor, the organic molecule FITC is used as the response signal for measuring pH, while AuNCs is used as the reference signal due to its good stability at different pH. As the LAMP reaction proceeds, the pH of the solution decreases, the green fluorescence of FITC decreases to varying degrees, while the red fluorescence of AuNCs remains basically unchanged. -4 ng / mL~10 2 ng / mL and FGreen / FRed value showed a good linear relationship (y=-0.12x+5.15, R 2 =0.9696), x is lg (ng / LAMP system), y is FGreen / FRed value, through Figure 4 The electrophoresis diagram verifies that when the nucleic acid concentration is 10 -2 ng / mL~10 2 ng / mL, there is a bright band. When the nucleic acid concentration is between 10 -3 ng / mL, there is a faint brightness band. -4 ng / mL, no specific band was seen, indicating that its detection limit LOD was approximately 7.4×10 -3 ng / mL (481CFU / g).

[0124] Example 4 Sample Detection

[0125] (1) Sample processing

[0126] Take 25 g of sample nuts, add them to 225 mL of sterilized water, shake for 10 min, take 200 μL of the shaken solution and add it to 100 mL of potato liquid culture medium, and culture at 28°C for 40 h.

[0127] (2) Sample DNA extraction

[0128] Take the mycelium in the potato liquid culture medium in step (1) and extract the sample genomic DNA according to the method in step (1) of Example 2.

[0129] (3) Establishment and detection of FITC@AuNCs-LAMP system for nut samples

[0130] 25 μL of LAMP reaction system, including 8 μL of 2×isothermal amplification buffer, 1.25 μL of dNTPs, 1 μL of Bst 2.0 DNA polymerase (Shanghai Shenggong Biotechnology Service Co., Ltd.), 0.6 μL of each of the outer primer and the inner primer, 0.3 μL of the loop primer, 1.25 μL of the mixed DNA template of Aspergillus, Penicillium and Fusarium extracted in step (2) of Example 4, and 7 μL of FITC@AuNCs, 3.5 μL sterile ultrapure water. The amplification procedure is: constant temperature at 63°C for 55 min, enzyme inactivation at 80°C for 5 min. After the LAMP reaction is completed, the LAMP product is taken to perform quantitative detection of the mold contained in the actual sample according to the standard curve in step (4) of Example 3.

[0131] In order to verify the practical applicability of this method, this method was compared with the traditional plate colony counting method. The results are shown in Table 1. There is no significant difference between the results of the two methods, indicating that this method can be used for actual sample detection and its results are reliable and accurate.

[0132] Table 1 Comparison of the results of analyzing nut samples by this method and the traditional plate colony counting method

[0133]

[0134] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0135] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention. SEQUENCE LISTING <110> Jiangnan University <120> A ratiometric fluorescence biosensor based on gold nanoclusters and its preparation method and its application in detecting Aspergillus Application in bacteria <130> 6 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 18 <212> DNA <213> (Artificial synthesis) <400> 1 gcagagggta ctttgggt 18 <210> 2 <211> 20 <212> DNA <213> (Artificial synthesis) <400> 2 ctctacttgt ggcgtatcgg 20 <210> 3 <211> 40 <212> DNA <213> (Artificial synthesis) <400> 3 ctttacacgg gcacggacac ctaatcgtgc cctggaacgg 40 <210> 4 <211> 41 <212> DNA <213> (Artificial synthesis) <400> 4 tcgacgagtc gagttggggt tttctccggc cagtatttag c 41 <210> 5 <211> 19 <212> DNA <213> (Artificial synthesis) <400> 5 ccaagacgta ggtctcacc 19 <210> 6 <211> twenty one <212> DNA <213> (Artificial synthesis) <400> 6 aatgcagcaa atcttgggtg g 21

Claims

1. A ratiometric fluorescence biosensor based on gold nanoclusters, characterized in that: The ratio fluorescence biosensor comprises a primer probe and FITC@AuNCs, wherein the primer probe comprises an upstream outer primer, a downstream outer primer, an upstream inner primer, a downstream inner primer, an upstream loop primer and a downstream loop primer; The nucleotide sequence of the upstream outer primer is shown in SEQ ID No.1, the nucleotide sequence of the downstream outer primer is shown in SEQ ID No.2, the nucleotide sequence of the upstream inner primer is shown in SEQ ID No.3, the nucleotide sequence of the downstream inner primer is shown in SEQ ID No.4, the nucleotide sequence of the upstream loop primer is shown in SEQ ID No.5, and the nucleotide sequence of the downstream loop primer is shown in SEQ ID No.6; The FITC@AuNCs were prepared by the following method: HAuCl4 and BSA solutions are mixed, and the pH of the reaction solution is adjusted to alkaline, and the reaction is stirred to obtain AuNCs; the obtained AuNCs are dissolved in a buffer solution to obtain an AuNCs solution; an alcohol solution of FITC is added to the AuNCs solution, and the mixture is stirred to obtain the FITC@AuNCs.

2. A kit, characterized in that: Comprising the ratiometric fluorescence biosensor as described in claim 1.

3. Use of the ratiometric fluorescent biosensor according to claim 1 and the kit according to claim 2 in the quantitative or qualitative detection of mold; The mold is selected from the genus Aspergillus, Penicillium or Fusarium; The Aspergillus molds are Aspergillus niger, Aspergillus flavus and Aspergillus jifengensis, the Penicillium mold is Penicillium viridans, and the Fusarium mold is Fusarium oxysporum.

4. The use according to claim 3, characterized in that: The application method comprises the following steps: mixing the primer probe, the FITC@AuNCs and the DNA template of Aspergillus fungus to carry out LAMP reaction, taking the LAMP product after the reaction is completed, and detecting the fluorescence intensity of the product.

5. The use according to claim 4, characterized in that: The LAMP reaction also includes isothermal amplification buffer, dNTPs, DNA polymerase, and water.

6. The use according to claim 3, characterized in that: The ratio fluorescence biosensor is used to detect Aspergillus fungi in nuts.

7. The use according to claim 6, characterized in that: The method for detecting Aspergillus mold in nuts comprises the following steps: S1: Add nuts containing Aspergillus fungi into sterilized water and shake them, add the shaken solution into culture medium for cultivation to obtain hyphae, and extract sample genomic DNA through the hyphae; S2: Perform LAMP reaction on the sample genomic DNA prepared in S1, the primer probe and the FITC@AuNCs solution, and after the reaction is completed, take the LAMP product for fluorescence detection.

Images