A method for detecting AFB1 based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology

Abstract

The present invention provides a preparation method and application of a fluorescent aptamer sensor based on a triple helix - padlock probe combined with double - cycle rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). The aptamer sensor described in the present invention includes a DNA triple - helix structure, a circular template strand, hairpin probes HP1 and HP2, wherein HP2 is a hairpin double - labeled with 6 - FAM and BHQ - 1. The DNA triple - helix structure is composed of an aptamer strand and a trigger strand. The trigger strand can initiate the RCA amplification reaction in the presence of the target aflatoxin B1 (AFB1), and further trigger the CHA reaction, enabling the originally quenched fluorescence signal to be restored. The AFB1 aptamer sensor described in the present invention cleverly utilizes the DNA triple - helix structure, combines the RCA - CHA cascade amplification reaction, effectively reduces the background signal of the reaction while doubling the amplified signal, thereby realizing the sensitive detection of AFB1 in food.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and specifically relates to a preparation method of a fluorescence aptamer sensor combining triple helix - padlock structure, rolling circle amplification and catalytic hairpin assembly double - cycle, and its application in detecting aflatoxin B1 in food. Background Art

[0002] Mycotoxins are a class of secondary metabolites produced by molds, which are widely distributed in nature and are very common in food contamination. Aflatoxin (AF) is a highly toxic secondary metabolite produced by the fungi Aspergillus flavus and Aspergillus parasiticus. It is reported that aflatoxin is a highly stable natural mycotoxin. Among them, aflatoxin B1 (AFB1) is the most toxic one among aflatoxins and has been listed as a class I carcinogen by the International Agency for Research on Cancer. AFB1 has varying degrees of harm to the liver, immune system and reproductive system of humans and several animals. To ensure public health and food safety, it is of great significance to strengthen the detection of AFB1 in food.

[0003] Existing AFB1 detection methods mainly include chromatographic analysis methods such as liquid chromatography, liquid chromatography - tandem mass spectrometry and gas chromatography - tandem mass spectrometry. Although these methods can achieve high - sensitivity detection of AFB1, most of these methods have problems such as high requirements for equipment and technical operations, complex sample processing, time - consuming and high costs, and are not suitable for large - scale food safety detection. Therefore, it is extremely important to construct a simple, efficient and suitable method for rapid detection to detect AFB1 in food.

[0004] Fluorescent aptamer sensors are based on aptamers for molecular recognition, using fluorescent groups as signal transducers to achieve rapid detection of the target analyte with high sensitivity and high selectivity. Aptamers are molecular probes that can specifically bind to target substances. Compared with antibodies, aptamers have the advantages of simple structure, easy synthesis and modification, wide adaptation range, easy preparation and storage. The triple helix molecular switch (THMS) is a special nucleic acid structure in which two arm segments of the aptamer chain bind to the primer chain sequence through Watson-Crick and Hoogsteen base pairing to form THMS. Compared with double helix DNA molecular switches and molecular beacon-based molecular structures, THMS has obvious advantages such as high sensitivity, good stability, selective retention and the original aptamer affinity. Rolling circle amplification (RCA) is a simple and effective isothermal DNA replication technique. On a circular DNA template, replication along the circular probe synthesizes large molecular weight tandem products, and RCA can generate thousands of repeated DNA nucleotides. Catalytic hairpin assembly (CHA) is an enzyme-free, highly efficient and isothermal amplification method. CHA has been widely used in the AFB1 detection system due to its advantages of simple control, easy operation and high detection sensitivity. The target long chain is amplified by RCA cycling to open a pre-designed hairpin, thus cleverly combining RCA and CHA to construct an RCA-CHA cascade amplification reaction, which can achieve double amplification of the fluorescence detection signal.

[0005] Combining the significant advantages of cascade amplification reactions, the present invention proposes a fluorescent biosensor based on THMS and RCA-CHA cascade amplification reactions for the rapid detection of AFB1 in food. This method uses THMS to replace the traditional form of aptamer and trigger chain binding, effectively reducing the false positives caused by unbound trigger chains. At the same time, THMS is combined with the RCA-CHA cascade amplification reaction, so that higher fluorescence signals can be obtained, improving the detection sensitivity. The present invention can quickly, sensitively and accurately detect AFB1 in food and has good application prospects. Summary of the Invention

[0006] The main object of the present invention is to provide a novel biosensor for detecting the content of AFB1 in food, which can be combined with RCA and CHA cascade amplification reactions to achieve cascade amplification of fluorescence signals during the detection of AFB1, facilitating the reduction of background signals and improving the sensitivity of the sensor.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] 1. A method for detecting AFB1 based on the combination of triple helix molecular switch and RCA-CHA cascade amplification technology is carried out according to the following steps:

[0009] (1) Preparation of the triple helix - padlock structure: Take the aptamer strand with a concentration of 100 μM and the primer strand with a concentration of 100 μM, add them into a sterilized centrifuge tube with 50 mM Tric - HCl buffer solution, incubate to assemble the triple helix - padlock structure, and store it in a low - temperature environment after preparation.

[0010] (2) Pretreatment of DNA strands H1 and H2: After H1 and H2 are activated and configured into a mother liquor with a concentration of 100 μM, they should be annealed before use to form a hairpin structure. The specific steps are as follows: Take 100 μM of H1 and H2 and add them into two sterilized centrifuge tubes respectively, then add 50 mM Tric - HCl buffer solution into each centrifuge tube, perform high - temperature annealing on the centrifuge tubes, and then let them stand to slowly cool to room temperature to obtain the reaction product, and store the reaction product in a low - temperature environment.

[0011] (3) Construction of the fluorescent aptamer sensor: First, the triple helix - padlock structure needs to be prepared in advance. When the sample contains AFB1, the aptamer strand can specifically bind to AFB1, thus opening the "padlock" structure and releasing the primer strand in the triple helix - padlock structure. The primer strand can circularize the circular template strand phosphorylated at the 5' end under the action of T4 DNA ligase to form a circular template, and initiate the RCA reaction under the action of phi29 DNA polymerase and dNTPs to amplify the target long strand. The amplification product of the RCA reaction further opens H1, exposing the complementary base sequences of H1 and H2, thus opening H2 labeled with the fluorescent group 6 - FAM and the quenching group BHQ - 1, forming a large number of H1 - H2 complex products. As FAM moves away from BHQ - 1, the fluorescence signal recovers. The H1 - H2 complex products break away from the RCA amplification strand, and the RCA reaction amplification strand can further open H1, enabling a double - cycle signal amplification of RCA - CHA to form in the system. By measuring the change in fluorescence intensity through fluorescence, a relationship curve between the fluorescence signal response value and the concentration of the target substance is established.

[0012] (4) Application in the detection of food samples: Measure the fluorescence signal of the unknown - concentration food sample to be detected through step (3), and substitute the detection result into the standard curve to obtain the concentration of AFB1 in the sample.

[0013] Further defined, in step (1), the incubation temperature of the triple helix - padlock structure is 37 °C, the incubation duration is 1 - 1.5 h, and the low - temperature storage environment for the prepared triple helix - padlock structure is 4 °C.

[0014] Further defined, in step (2), the water - bath temperature for annealing is 95 °C, the water - bath duration is 10 min, and the low - temperature storage environment for the reaction product is 4 °C.

[0015] Further limitation: in step (3), the shaking incubator temperature for adding the target to be measured is 37°C, and the incubation duration is 1 - 1.5 h; the circular template strand is phosphorylated at the 5' end in advance and forms a circular template under the action of T4 DNA ligase, with an incubation temperature of 22°C and an incubation duration of 3 h.

[0016] Further limitation: in step (3), the phi29 DNA polymerase system solution added includes Phi29 DNA polymerase, 1X Phi29 buffer, dNTPs, with an incubation temperature of 30°C and an incubation duration of 15 - 90 min, and a DNA long chain with a specific sequence is generated by driving rolling circle amplification with Phi29 DNA polymerase.

[0017] Further limitation: the aptamer strand sequences in steps (1), (2), and (3) are one of 5'-CTC TCT TGC ACG TGTTGT CTC TCT GTG TCT CGT GCT CTC TC-3', 5'-CTC TCT CTG CAC GTG TTG TCT CTC TGTGTC TCG TGC CTC TCT C-3', 5'-CTC TCT CTT GCA CGT GTT GTC TCT CTG TGT CTC GTGCTC TCT CTC-3', or 5'-CTC TCT CTC TGC ACG TGT TGT CTC TCT G TG TCT CGT GCC TCTCTC TC-3'; the primer strand sequence is 5'-GAG AGA GAG AGA GAC GAG CAG AGT TAGGGT T-3'; the circular template strand sequence is 5'-P-ACT CTG CTC GCC CTA TCC CTA ACC CTA-3'; the hairpin probe strand H1 sequence is 5'-CCC TAA CCC TAA CTC TCA AGG GTA GGG CGG GTC AGA GTTAGG G-3'; the DNA strand H2 sequence is 5'-FAM-AAC TCT ACC CGC CCT ACC CTT GAG AGT TAGGGA GGG TAG GGC GGG T-BHQ-1-3'.

[0018] Compared with the prior art, the present invention has the following remarkable advantages:

[0019] 1. The present invention utilizes a precisely designed triple-helix molecular switch to ingeniously form a triple-stranded body-locked structure by base complementary pairing and Hoogsteen base pairing between the aptamer strand of AFB1 and the primer strand capable of initiating rolling circle amplification. Compared with the double-helix DNA molecular switch, the triple-stranded body-locked structure has advantages such as high sensitivity, good stability, selective retention, and the affinity of the original aptamer.

[0020] 2. The present invention utilizes a specially designed circular template strand sequence to enable the rolling circle amplification to restore the signal of the fluorescent group through a series of reactions, generating a large number of H1-H2 complexes. Subsequently, the amplified long template strand is released and can further open the DNA strand H1, thereby realizing the RCA-CHA double cycle and significantly improving the sensitivity of the sensor.

[0021] 3. Based on the sensing strategy of the present invention, it has advantages such as good specificity, high accuracy, good repeatability, and good stability, which is conducive to the popularization and application of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation method of the fluorescent aptamer sensor and the detection of AFB1.

[0023] Figure 2 Fluorescence intensity of the system of the sensor constructed in Example 1 of the present invention in the absence of AFB1 (dotted line), in the presence of 5 ng / mL AFB1 (dashed line), and 50 ng / mL AFB1 (solid line).

[0024] Figure 3 Selectivity comparison diagram of the fluorescent aptamer sensor constructed in Example 1 of the present invention for AFB1 in the presence of other interfering toxins. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following combines the drawings and examples to further describe in detail the specific implementation manners of the present invention. The following examples are used to illustrate the present invention but not to limit the scope of the present invention.

[0026] EXAMPLE

[0027] A method for detecting AFB1 based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology, and its implementation method is as Figure 1 shown.

[0028] A method for detecting AFB1 based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology, comprising the following steps:

[0029] (1) Pretreatment of DNA strands H1 and H2: Centrifuge H1 and H2 at a speed of 4000 r / min for 5 min to prevent dispersion. Then open the lid of the tube, and according to the instruction manual, add different volumes of sterilized 50 mM Tris-HCl to the tube to prepare a 100 μM mother liquor, and vortex thoroughly to mix evenly. The above DNA strands should be annealed before use to form a hairpin structure. The specific steps are as follows: Add 5 μL of H1 strand with an initial concentration of 100 μM and 5 μL of H2 strand with an initial concentration of 100 μM to two sterilized centrifuge tubes respectively. Then, add 495 μL of 50 mM Tris-HCl buffer solution to each centrifuge tube and vortex thoroughly to mix evenly. Subsequently, perform high-temperature annealing on the centrifuge tubes. The annealing water bath temperature is 95 °C, and the water bath duration is 10 min. Then let it stand to slowly cool to room temperature to obtain the reaction product, and store the reaction product in a low-temperature environment. The storage environment temperature is 4 °C.

[0030] (2) Preparation of the triple helix-padlock structure: Add the aptamer strand with a concentration of 100 μM, the primer strand with a concentration of 100 μM, and 50 mM Tric-HCl buffer solution to a sterilized centrifuge tube for incubation. The incubation temperature is 37 °C, and the incubation duration is 1.5 h to assemble the triple helix-padlock structure. Then store it in a low-temperature environment. The storage environment temperature is 4 °C; among them, the volumes of the selected aptamer strand, primer strand, and 50 mM Tric-HCl are 10 μL, 10 μL, and 480 μL respectively.

[0031] (3) Construction of the fluorescent aptamer sensor: Add the triple helix-padlock structure prepared in step (3) to the target to be measured and incubate it on a shaker. The incubation environment is 1.5 h and 37 °C. Subsequently, place the circular template strand pre-labeled with a phosphate group, and the H1 strand and H2 strand treated in step (2) in a sterilized buffer solution, and mix thoroughly with a turbine to prepare a biosensor. Among them, the concentration of the triple helix-padlock structure is 2 μM, and the volume is 20 μL; the concentration of the circular template strand is 1 μM, and the volume is 10 μL; the concentrations of the H1 strand and H2 strand are 1 μM, and the volumes are 10 μL; in addition, the volume of T4 DNA ligase (5 U / μL) is 1 μL, the volume of Phi29 DNA polymerase (10 U / μL) is 0.5 μL, and the volume of dNTPs (10 mM) is 2 μL. Measure the change in fluorescence intensity by fluorescence to establish a relationship curve between the fluorescence signal response value and the target concentration.

[0032] (4) In the above example, the primer chain can hybridize with the stem sequence of the adapter chain to form a triplex-lock structure; the primer chain can circularize the phosphorylated circular template chain to form a circular template under the action of T4 DNA ligase, and further initiate the RCA reaction under the action of phi29 DNA polymerase and dNTPs to amplify the target long chain. The product of the RCA reaction further opens H1, exposing the bases of the complementary sequence of DNA chains H1 and H2, thereby opening H2 labeled by the fluorescent group 6-FAM and the quenching group BHQ-1. As FAM moves away from BHQ-1, the fluorescence signal is restored, forming a large amount of H1-H2 complex products, breaking away from the RCA amplification chain. The RCA amplification chain can further open the hairpin probe H1, so that a double-cycle signal amplification of RCA-CHA is formed in the system. The change in fluorescence intensity is measured by a fluorometer, and a relationship curve between the fluorescence signal response value and the target concentration is established.

[0033] (5) Application to food sample detection: The fluorescence signal of the food sample with unknown concentration is measured in step (5). The detection is not applicable to human or animal samples. The detection result is put into the standard curve to obtain the AFB1 concentration in the sample. Figure 2 Shown are the fluorescence intensities of the sensor constructed in Example 1 of the present invention in the absence of AFB1 (dotted line), the presence of 5 ng / mL AFB1 (dashed line), and 50 ng / mL AFB1 (solid line).

[0034] To further illustrate the present invention, the biosensor prepared by the present invention is used to detect AFB1.

[0035] Example

[0036] A method and application for detecting AFB1 based on a triple helix molecular switch combined with RCA-CHA cascade amplification technology, comprising the following steps:

[0037] In order to verify that the prepared fluorescent aptamer sensor based on the triple-helix molecular switch combined with RCA-CHA cascade amplification technology has specific recognition of AFB1, an AFB1 standard was added to a 50mM Tric-HCl buffer solution to make the concentration of AFB1 in the sample 50ng / mL; 50mM Tric-HCl buffer was used to prepare other interfering toxins (OTA, AFB2, FB1, ZEN) standard solutions, all with a concentration of 500ng / mL. The signal intensity corresponding to the vertical axis is the difference between the different targets and the blank sample at 520nm. The above-mentioned several different interfering toxin standards were detected according to the detection system constructed in Example 1, and the detection results are shown as follows: Figure 3 As shown, it is shown that the method of the present invention has high selectivity for AFB1.

[0038] Example

[0039] A method for detecting AFB1 based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology and its application, comprising the following steps:

[0040] (1) Actual sample treatment: Take 0.5 g of food spiked sample, add it to 10 mL of methanol / water solution (ratio 7:3), place it on a shaker and shake for 20 minutes, then take it out and centrifuge it in a centrifuge for 10 min at a rotation speed of 10,000 r / min. After centrifugation, take out the supernatant to obtain the food sample extract.

[0041] (2) Sample detection: Measure the fluorescence intensity signal according to the steps of Example 1, and substitute it into the standard curve to obtain the concentration of AFB1 in the sample.

[0042] (3) When using black tea as the actual sample for determination, based on the addition amount of 5 ng / mL, add AFB1 standard products with 0.1 times and 10 times of the reference amount to black tea respectively. Take 10 μL of the sample solution, measure the fluorescence intensity signal according to steps (1)-(5) of Example 1, substitute it into the standard curve detected in Example 1 to obtain the concentration of AFB1 in the sample. Each sample is measured 3 times repeatedly and the average value is taken, and the RSD value and recovery rate are calculated as shown in the following table:

[0043]

[0044] It is verified that the prepared fluorescence aptamer sensor has the characteristics of high accuracy, good sensitivity, good reproducibility and stability for the detection of AFB1. At the same time, the detection results of actual samples (such as black tea) show that the prepared sensor has very good practical application value.

[0045] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0046] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting AFB1 in food based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology, characterized in that, Including the following steps: (1) Preparation of the triple helix - padlock structure: Add the aptamer strand and the primer strand into a sterilized centrifuge tube, add the Tric-HCl buffer solution for incubation at a constant temperature to assemble the triple helix - padlock structure. After preparation, store it in a low-temperature environment. The aptamer strand sequence of the triple helix - padlock structure is one of SEQ ID NO.1: 5’-CTC TCT TGC ACG TGTTGT CTC TCT GTG TCT CGT GCT CTC TC -3’; SEQ ID NO.2: 5’-CTC TCT CTG CAC GTG TTGTCT CTC TGT GTC TCG TGC CTC TCT C -3’; SEQ ID NO.3: 5’-CTC TCT CTT GCA CGT GTTGTC TCT CTG TGT CTC GTG CTC TCT CTC-3’; SEQ ID NO.4: 5’-CTC TCT CTC TGC ACG TGTTGT CTC TCT GTG TCT CGT GCC TCT CTC TC-3’, and the primer strand sequence is as shown in SEQ IDNO.5: specifically 5’-GAG AGA GAG AGA GAC GAG CAG AGT TAG GGT T-3’; (2) Pretreatment of DNA strands H1 and H2: H1 and H2 are activated and configured into a mother liquor. Before use, they should be annealed to form a hairpin structure. The DNA strand H1 sequence is 5’-CCC TAA CCC TAA CTC TCA AGG GTA GGG CGG GTCAGA GTT AGG G-3’; the DNA strand H2 sequence is 5’-FAM-AAC TCT ACC CGC CCT ACC CTT GAGAGT TAG GGA GGG TAG GGC GGG T-BHQ-1-3’. The specific steps are as follows: Take H1 and H2 and add them into two sterilized centrifuge tubes respectively. Add the Tris-HCl buffer solution into each centrifuge tube, perform high-temperature annealing on the centrifuge tubes, and after annealing, let it stand and slowly cool to room temperature to obtain the reaction product, and store the reaction product in a low-temperature environment; (3) Construction of the fluorescent aptamer sensor: Prepare the triple helix-padlock structure in advance. When AFB1 is present in the sample, the aptamer strand specifically binds to AFB1, opening the "padlock" structure and releasing the primer strand in the triple helix-padlock structure for initiating the circularized 5'-phosphorylated circular template strand. The sequence of the circular template strand is shown as SEQ ID NO.6: specifically 5'-P-ACT CTG CTC GCC CTA TCC CTA ACC CTA-3'. Add the Phi29 DNA polymerase system solution and the treated DNA strands H1 and H2, vortex and mix well to prepare the aptamer sensor.

2. The method for detecting AFB1 in food based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology according to claim 1, wherein, The ratio of the aptamer strand to the primer strand is 1:1; the incubation time for preparing the triple helix-padlock structure is 1 to 1.5 h.

3. A method for detecting AFB1 in food based on a triple-helix molecular switch combined with RCA-CHA cascade amplification technology according to claim 1, characterized in that, The Phi29 DNA polymerase system solution includes Phi29 DNA polymerase, 1X Phi29 buffer, dNTPs; the incubation temperature is 30 °C, and the incubation duration is 15 to 90 min. The rolling circle amplification is driven by the Phi29 DNA polymerase and dNTPs fuel to generate a long DNA strand with a specific sequence.

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