Preparation method of a fluorescence aptamer sensor for simultaneous detection of ochratoxin A and aflatoxin B1

Abstract

The present invention relates to a method for preparing a fluorescence aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1, comprising the following steps: preparing a DNA double-labeled strand by using a PCR annealing program. The sensor is formed by hybridizing a DNA strand S1 labeled with a quenching group at the 3' end, a DNA strand S2 labeled with a quenching group at the 5' end, and a DNA strand S3 modified with fluorescent groups at both the 3' and 5' ends. According to the change of the response signal, a fluorescence aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 is obtained. Compared with other electrochemical sensors for detecting ochratoxin A and aflatoxin B1, the prepared fluorescence aptamer sensor has the advantages of high sensitivity, good repeatability, and high accuracy.

Description

Technical Field

[0001] The present invention relates to a method for preparing a fluorescence aptamer sensor for simultaneous detection of ochratoxin A and aflatoxin B1, and particularly relates to a method for preparing a DNA double-labeled strand. Background Art

[0002] During the growth period of grains and the storage process after production, due to the influence of environmental factors, grains are often easily contaminated by various fungi. Therefore, the possibility of coexistence of mycotoxins in grains and their derivatives is very high. Literature reports have found that ochratoxin A (OTA) and aflatoxin B1 (AFB1) usually coexist naturally in wheat and corn. At present, there are many research reports that the potential risk of coexistence of OTA and AFB1 may cause greater toxicity due to cumulative or synergistic effects, posing a huge threat to public health, especially infants and young children. Related studies have also found that OTA and AFB1 have synergistic cytotoxic effects. In addition to having nephrotoxicity and cytotoxicity, AFB1 also has a carcinogenic promoting effect, enhancing the genotoxicity and carcinogenicity of OTA. Therefore, there is an urgent need to develop a method that can achieve simultaneous detection in a single analytical operation to accurately evaluate the contamination levels of OTA and AFB1 in food matrices. Currently, many methods for determining AFB1 and OTA have been developed. The traditional thin-layer chromatography method has a large specific workload for processing analytical samples and low accuracy in quantitative detection. High-performance liquid chromatography (HPLC) or high-performance liquid chromatography-mass spectrometry (MS) methods have disadvantages such as high equipment usage and maintenance costs, high operation requirements, and long analysis times. Although immunoassay methods can meet the requirements of sensitivity, selectivity, and convenience, the properties, synthesis, and retention period of antibodies limit the application of antibodies in analytical methods. Colorimetric sensors have defects in stability and sensitivity, while electrochemical sensor methods have poor stability and repeatability due to frequent modification of the electrode surface and interference from the sensing efficiency of nanomaterials. In summary, the fluorescence sensor method based on nucleic acid aptamers has relatively significant advantages, such as strong specificity, simple operation, and good stability, and has quite good application prospects. Summary of the Invention

[0003] A method for preparing a fluorescence aptamer sensor for simultaneous detection of ochratoxin A and aflatoxin B1 is carried out according to the following steps:

[0004] (1)Preparation of DNA double-labeled strand: It is formed by connecting DNA strand S1 containing 23 bases and DNA strand S2 containing 26 bases to DNA strand S3 through base complementary pairing; Take 1-2 μM of each of DNA strands S1, S2, and S3, 10-20 μL each, into a 100-200 μL centrifuge tube, vortex thoroughly to mix evenly, place it in a gradient PCR instrument, and perform an annealing program at an annealing temperature of 95-100 °C to finally obtain a DNA double-labeled strand of DNA strand S1 labeled with a quenching group at the 3' end, DNA strand S2 labeled with a quenching group at the 5' end, and DNA strand S3 labeled with fluorescent groups at both the 3' and 5' ends; The sequence of DNA strand S1 is 5'-GACACAGAGAGACAACACGTGCA-3', and the quenching group labeled at the 3' end is one of BHQ1, BHQ2, TAMRA, Dabcyl; The sequence of DNA strand S2 is 5'-TCCCTTTACGCCACCCACACCCGATC-3', and the quenching group labeled at the 5' end is one of BHQ1, BHQ2, TAMRA, Dabcyl; The sequence of DNA strand S3 is 5'-AATGGATGTTGTCTCTCTGT20CAGATAGTGTGGTGTAAAGGGA-3', and the fluorescent groups labeled at the 3' and 5' ends are respectively one of Cy3, Cy5, FAM, ROX, Alexa Fluor 488, Alexa Fluor 594. The fluorescent groups labeled at the 3' and 5' ends of DNA strand S3 have different colors and different maximum emission wavelengths under the excitation wavelength of the fluorescence spectrum.

[0005] (2)The fluorescent aptamer sensor is formed by hybridization of DNA strand S1 labeled with a quenching group at the 3' end, DNA strand S2 labeled with a quenching group at the 5' end, and DNA strand S3 modified with fluorescent groups at both the 3' and 5' ends; When the sample contains the target substances ochratoxin A and aflatoxin B1, the aptamer strand binds to the target substances, causing the fluorescent group and the quenching group to approach each other, generating a strong FRET effect and resulting in a change in the response signal; Thus, a fluorescent aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 is obtained.

[0006] Further defined, in step (1), the annealing program is to first raise the annealing temperature to 95-100 °C for 5-10 min, and then slowly cool down to 4-10 °C and maintain for 30-45 min.

[0007] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines with the attached drawings to describe in detail as follows. Description of the Drawings

[0008] Figure 1 Shows the agarose gel electrophoresis characterization diagram of a fluorescence aptasensor for simultaneous detection of ochratoxin A and aflatoxin B1;

[0009] Figure 2 Shows the schematic diagram of a fluorescence aptasensor for simultaneous detection of ochratoxin A and aflatoxin B1. Detailed implementation manners

[0010] The following combines the accompanying drawings and embodiments to further describe in detail the specific implementation manners of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention. Embodiment 1

[0011] The specific steps are as follows:

[0012] (1) Preparation of DNA double-labeled strand: It is formed by connecting DNA strand S1 containing 23 bases and DNA strand S2 containing 26 bases to DNA strand S3 by base complementary pairing. Pipette 20 μL each of 2 μM DNA strands S1, S2, and S3 into a 100 μL centrifuge tube, vortex and mix well, and place it in a gradient PCR instrument to execute the annealing program. The preset thermal denaturation and annealing temperature program is as follows: 5 min at 95 °C, and then slowly cool down to 4 °C and keep for 30 min. Finally, a DNA double-labeled strand is obtained in which the 3' and 5' ends of DNA strand S3 are labeled with Cy3 and Cy5 respectively, the 3' end of DNA strand S1 is labeled with BHQ1, and the 5' end of DNA strand S2 is labeled with BHQ2;

[0013] (2) Agarose gel electrophoresis characterization: Use 3.0% agarose gel to characterize the preparation of the sensor. The electrophoresis results are as Figure 1 shown. The band in lane 1 represents DNA strand S1, the band in lane 2 represents DNA strand S2, the band in lane 3 represents DNA strand S3, and the band in lane 4 represents the DNA double-labeled strand composed of DNA strand S1, DNA strand S2, and DNA strand S3.

[0014] (3) The fluorescence aptasensor is formed by hybridization of DNA strand S1 labeled with BHQ1 at the 3' end, DNA strand S2 labeled with BHQ2 at the 5' end, and DNA strand S3 modified with Cy3 and Cy5 at the 3' and 5' ends respectively. When the sample contains the target substances ochratoxin A and aflatoxin B1, the aptamer strand binds to the target substances, causing the fluorophore and the quencher group to approach each other, generating a strong FRET effect and causing a change in the response signal. Thus, a fluorescence aptasensor for simultaneous detection of ochratoxin A and aflatoxin B1 is obtained. Embodiment 2

[0015] The specific steps are as follows:

[0016] (1)Preparation of DNA double-labeled strand: It is formed by connecting DNA strand S1 containing 23 bases and DNA strand S2 containing 26 bases to DNA strand S3 through base complementary pairing. Pipette 10 μL each of 1 μM DNA strands S1, S2, and S3 into a 100 μL centrifuge tube, vortex thoroughly to mix evenly, and place it in a gradient PCR instrument to execute the annealing program. The preset thermal denaturation and annealing temperature program is as follows: 10 min at 95 °C, and then slowly cool down to 4 °C and hold for 45 min. Finally, a DNA double-labeled strand is obtained, where the 3' and 5' ends of DNA strand S3 are labeled with FAM and ROX respectively, the 3' end of DNA strand S1 is labeled with TAMRA, and the 5' end of DNA strand S2 is labeled with Dabcyl;

[0017] (2)The fluorescence aptamer sensor is formed by hybridizing DNA strand S1 labeled with TAMRA at the 3' end, DNA strand S2 labeled with Dabcyl at the 5' end, and DNA strand S3 modified with FAM and ROX at the 3' and 5' ends respectively. When the sample contains the target substances ochratoxin A and aflatoxin B1, the aptamer strand binds to the target substances, causing the fluorophore and the quencher to approach each other, generating a strong FRET effect, resulting in a change in the response signal, and obtaining a fluorescence aptamer sensor for simultaneous detection of ochratoxin A and aflatoxin B1.

[0018] The prepared fluorescence sensor has the characteristics of high accuracy, wide linear range (OTA is 0.05 - 20 ng / mL, AFB1 is 0.08 - 10 ng / mL), and low detection limit (OTA is 0.029 ng / mL, AFB1 is 0.035 ng / mL) for the detection of ochratoxin A and aflatoxin B1. At the same time, the detection results of actual samples (such as red wine and corn) show that the prepared sensor has very good practical application value.

[0019] 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 the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. 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 claims involved.

[0020] 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 preparation method of a fluorescence aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1, characterized in that, Including the following steps: (1) Preparation of DNA double-labeled strand: It is formed by connecting DNA strand S1 containing 23 bases and DNA strand S2 containing 26 bases to DNA strand S3 through base complementary pairing; take 1 - 2 μM of each of DNA strands S1, S2, and S3, 10 - 20 μL each, into a 100 - 200 μL centrifuge tube, vortex thoroughly to mix evenly, and place it in a gradient PCR instrument to perform the annealing program, finally obtaining a DNA double-labeled strand with a quenching group labeled at the 3' end of DNA strand S1, a quenching group labeled at the 5' end of DNA strand S2, and a DNA strand S3 with both the 3' and 5' ends labeled with fluorescent groups; the sequence of the DNA strand S1 is 5'-GACACAGAGAGACAACACGTGCA-3', and the quenching group labeled at the 3' end is one of BHQ1, BHQ2, TAMRA, Dabcyl; the sequence of the DNA strand S2 is 5'-TCCCTTTACGCCACCCACACCCGATC-3', and the quenching group labeled at the 5' end is one of BHQ1, BHQ2, TAMRA, Dabcyl; the sequence of the DNA strand S3 is 5'-AATGGATGTTGTCTCTCTGT20CAGATAGTGTGGTGTAAAGGGA-3', and the fluorescent groups labeled at the 3' and 5' ends are respectively one of Cy3, Cy5, FAM, ROX, Alexa Fluor 488, Alexa Fluor 594. The fluorescent groups labeled at the 3' and 5' ends of the DNA strand S3 have different colors and different maximum emission wavelengths under the excitation wavelength of the fluorescence spectrum. (2) The fluorescence aptamer sensor is formed by hybridization of a DNA strand S1 with a quenching group labeled at the 3' end, a DNA strand S2 with a quenching group labeled at the 5' end, and a DNA strand S3 with both the 3' and 5' ends modified with fluorescent groups; when the sample contains the target substances ochratoxin A and aflatoxin B1, the aptamer strand binds to the target substances, causing the fluorescent group and the quenching group to approach each other, generating a strong FRET effect and resulting in a change in the response signal; thus, a fluorescence aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 is obtained.

2. The preparation method of the fluorescence aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 according to claim 1, characterized in that, In step (1), the annealing program is to first raise the annealing temperature to 95 - 100 °C for 5 - 10 min, and then slowly cool down to 4 - 10 °C and maintain for 30 - 45 min.

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