CN109946274B - Method for detecting aflatoxin B1 based on aptamer inherent conformation induction - Google Patents
Method for detecting aflatoxin B1 based on aptamer inherent conformation induction Download PDFInfo
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Abstract
The invention discloses a method for detecting aflatoxin B based on aptamer inherent conformation induction1The method of (1) labeling aflatoxin B by using TAMRA fluorescent molecule1（Aflatoxin B1,AFB1) The aptamer is added with EvaGreen molecules which can specifically recognize and embed into a DNA double-stranded structure to emit light, and based on a Fluorescence Resonance Energy Transfer (FRET) mechanism, through the change of the inherent conformation response of the aptamer molecules to target molecules, the fluorescence color development of the double-stranded dye EvaGreen is combined, so that the aptamer can emit light to AFB (atomic fluorescence resonance spectroscopy)1Rapid homogeneous quantitation was performed. The method disclosed by the invention only utilizes the self structure of the aptamer, avoids the complex design of a nucleic acid probe, and on the other hand, all reactions are carried out in one tube at room temperature by one mixing step, so that the steps of separation and temperature control are eliminated. The method has the advantages of simple principle, low cost, high sensitivity, good selectivity and the like, and can be used for AFB1Has a detection limit of 0.32ng/ml, can detect samples of different food substrates, and is a competitive AFB1A rapid detection method.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting aflatoxin B based on aptamer inherent conformation induction1The method of (1).
Background
Aflatoxin B1（Aflatoxin B1, AFB1) Is one of the most important mycotoxins affecting the quality of agricultural products and is a serious threat to food safety. AFB1Is prepared from Aspergillus flavusAspergillus flavus) And Aspergillus parasiticus (A), (B)Aspergillus parasiticus) The secondary metabolites produced by these fungi are recognized as class I carcinogens by the International agency for research on cancer (IARC) and are considered to be one of the major causes of liver cancer. To date, AFB1The mycotoxin is found to be the mycotoxin with the strongest toxicity, and has the advantages of more stable physicochemical property, high temperature resistance, difficult removal and the toxicity which is about 68 times stronger than that of arsenic. Therefore, AFB is tightly controlled around the world1And (4) pollution. For example, AFB is currently found in Chinese and American foods1Maximum allowable level of20 ug/kg, 8 ug/kg in the European Union. AFB1Can be produced in improperly stored food products, including crops (e.g., corn) and their processed products (e.g., peanut oil).
For AFB1The commonly available techniques for analytical detection are mainly liquid chromatography-mass spectrometry (LC-MS) and High Performance Liquid Chromatography (HPLC), although these techniques lend themselves to AFB1Assays have high reliability, sensitivity and accuracy, but they still suffer from limitations such as requiring expensive equipment, specialized technicians and long analysis costs, which prevent their field assay applications. By way of comparison, immunoassays based on the specific binding of antibodies and antigens will contribute to AFB1High sensitivity and rapid quantitative detection while avoiding the need for expensive instrumentation. However, the dependence on antibodies has hindered their widespread use due to their poor stability, laborious preparation, high antibody costs, and the like.
Taking into account AFB1Severe toxicity and prevalence in food, more simple, sensitive, and rapid development for detecting AFB1The method of (3) is essential for food safety control. The advent of an artificial nucleic acid affinity reagent (aptamer) for the development of AFB1The new analytical methods of (a) provide unprecedented opportunities. Aptamers have better thermal and chemical stability compared to antibodies, in particular the programmability and structure-switching properties of nucleic acids, allowing the design of various types of nucleic acid probes and the ability to construct homogeneous assays for target detection, thereby eliminating separation and washing processes. Thus, AFB is detected in the field1In the case of (3), it is preferable to construct an assay method for rapid detection using an aptamer.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin B based on aptamer inherent conformation induction1The method of (1). The specific technical scheme is as follows:
AFB (active carbon boron)1The nucleotide sequence of the aptamer is shown as SEQ ID No.1 in the sequence table;
the AFB1The aptamer has a secondary structure as shown in figure 1;
the AFB1The aptamer was labeled with TAMRA fluorescent molecule at its 5' end.
The aptamer is used for detecting AFB1The method comprises the following steps:
(a) preparation of Aflatoxin B1A standard curve of concentration versus fluorescence intensity;
(b) detecting the sample to be detected according to the process of preparing the standard curve to obtain the fluorescence intensity of the sample to be detected, and calculating the aflatoxin B in the sample to be detected according to the standard curve1The concentration of (c).
Wherein, the step of preparing the standard curve comprises the following steps:
(1) adding fluorescent dye and AFB (fluorescent dye) for specifically recognizing double-stranded DNA (deoxyribonucleic acid) into a buffer solution system1Aptamer to prepare AFB1Detecting the liquid;
the buffer system comprises: ultrapure water, 33 mM Tris-HCl (pH 7.9 at 25 ℃), 10 mM MgCl2, 66 mM KCl；
The fluorescent dye is an EvaGreen molecule which is embedded into a DNA double-chain structure and emits light;
the aptamer is marked with TAMRA fluorescent molecules at the 5' end, and the nucleotide sequence of the aptamer is shown as SEQ ID No.1 in a sequence table.
(2) In AFB1Adding AFB with different concentrations into the detection solution1Mixing the standard solution uniformly and reacting;
(3) measuring the fluorescence intensity of the reaction mixed solution obtained in the step (2) to obtain the fluorescence intensity along with AFB1Standard curve of concentration change.
The step (1) comprises the following steps: combining a fluorescent dye with AFB1Adding the aptamer into the buffer solution, and incubating for 0-20 min at 15-35 ℃ to obtain AFB1And (6) detecting the liquid.
The step (2) comprises the following steps: the prepared AFB to be tested1And (3) adding the standard solution into the solution obtained in the step (1), and reacting at 15-35 ℃ for more than 25 min.
The reaction of step (3) was incubated at 37 ℃ for 35 min.
The fluorescence detection conditions in the step (4) are that the excitation wavelength is 488nm, the emission wavelength range is 518-700nm, and the scanning step length is 1 nm.
The basic principle on which the invention is based is as follows: as shown in FIG. 2, in AFB1The 5' end of the aptamer is marked with a TAMRA fluorescent molecule, the aptamer forms a specific secondary structure through base complementary pairing self-hybridization, and an organic fluorescent dye EvaGreen which can specifically recognize and be embedded into a DNA double-chain structure to emit light is introduced. Due to the high overlap of the excitation spectrum of the EvaGreen molecule and the emission spectrum of the TAMRA molecule, the two molecules will undergo efficient Fluorescence Resonance Energy Transfer (FRET) at close distances. Then introducing AFB1The original structure of the aptamer is changed due to the specific combination with the aptamer, the DNA double-chain structure is reduced, the fluorescence intensity of EvaGreen is reduced, the distance between the EvaGreen molecule and TAMRA molecule is reduced, the FRET effect is increased, the fluorescence intensity of the EvaGreen molecule is further reduced, and the reduction of the fluorescence intensity of the EvaGreen molecule also reduces the fluorescence intensity of the TAMRA molecule. Finally, the product of the fluorescence intensities of the two fluorescent molecules is used as a signal to output to AFB1Quantitative analysis of (3).
Compared with the prior art, the invention has the following advantages:
1. the invention provides a method for detecting aflatoxin B based on aptamer inherent conformation induction1The method involving AFB only1The internal structure of the aptamer can avoid the complex design of a nucleic acid probe.
2. The detection method provided by the invention is AFB due to FRET mechanism1The aptamer has a synergistic response of conformational and distance change to a target molecule, so that the detection signal-to-noise ratio of the aptamer is remarkably improved, and the detection sensitivity of the method is greatly improved.
3. The detection method provided by the invention has the advantages that under the room temperature condition, the whole process only needs to be carried out once homogeneous mixing reaction in one test tube, so the detection process is quite simple, and the separation and temperature control processes are omitted.
4. The detection method provided by the invention is suitable for detecting AFB in various complex food matrixes (peanut oil, broad bean paste, corn and the like)1。
5. The detection method provided by the invention has practical universality and design simplicity for other food pollutants such as ochratoxin A, aflatoxin M1, terramycin, staphylococcus aureus and the like by using different aptamers.
Drawings
FIG. 1 is AFB1Schematic representation of the initial secondary structure of the aptamer.
FIG. 2 is a diagram of the method provided by the present invention to detect AFB1Schematic diagram of the principle of (1).
FIG. 3 is a standard curve plotted in example 2.
FIG. 4 is a graph of the detection of AFB in peanut oil, soybean paste and corn meal extracts of example 31The result of (1).
FIG. 5 is the results of the selectivity test for other mycotoxins in example 4.
FIG. 6 is an AFB assay in comparative example 11Schematic diagram of the principle of (1).
Detailed Description
Example 1
Aflatoxin B detection based on aptamer inherent conformation induction1The method specifically comprises the following steps
（1）AFB1Preparation of detection liquid:
in a buffer system (33 mM Tris-HCl (pH 7.9 at 25 ℃), 10 mM MgCl266 mM KCl) was added 3.5ul of 20x EvaGreen and 3.5ul of 10uM AFB1Diluting aptamer with ultrapure water to 31.5ul, mixing, and reacting at 25 deg.C for 5min to obtain AFB1And (6) detecting the liquid.
The aptamer is marked with TAMRA fluorescent molecules at the 5' end, and the nucleotide sequence of the aptamer is shown as SEQ ID No.1 in a sequence table;
(2) measurement method
In AFB13.5ul of AFB to be detected is added into the detection solution1And uniformly mixing the solution, incubating the solution at 25 ℃ for 40min, and then carrying out fluorescence intensity determination on the basis that the fluorescence detection condition is that the excitation wavelength is 488nm and the emission wavelength range is 518-700 nm.
Example 2
AFB1Preparation of a Standard Curve relating concentration to fluorescence intensity
The detection procedure of example 1 was used, in which the AFB to be detected1The solution is AFB1Standard solutions (0, 1, 10, 50, 100, 200, 300 ng/ml) were then recorded for fluorescence intensity at emission wavelengths of 530nm and 582nm, and the product of the two fluorescence intensities was calculated and analytically fitted to AFB1Reasonable standard curve of concentration versus fluorescence intensity product value (fig. 3): y = -17.32x + 10730, R2= 0.9972, and the detection limit of AFB1 was calculated to be 0.32 ng/ml.
Example 3
Detection of food samples
(1) 5g of the sample (thick broad-bean paste, peanut oil and corn flour), 20mL of the methanol/water (70: 30, v/v) mixed solution was added to a 50mL centrifuge tube and after gentle shaking on a shaker for 1h, the centrifuge tube was centrifuged at 6000rpm for 10min at room temperature. Then taking the supernatant, diluting the supernatant by 10 times with water, and uniformly mixing the supernatant for later use;
(2) preparing AFB with the concentration of 100 ng/ml and 300ng/ml by using the solution obtained in the step (1)1A sample solution;
(3) the detection procedure of example 1 was used, in which the AFB to be detected1The solution is AFB prepared in the step (1)1The sample solution, then the fluorescence intensities at the emission wavelengths of 530nm and 582nm were recorded and the product of the two fluorescence intensities was calculated and substituted into the standard curve obtained in example 2: y = -17.32x + 10730, R2= 0.9972, obtaining AFB in sample solution to be detected1The results of concentration are shown in FIG. 4, and the recovery rate is 89.2% -110.8%.
Example 4
Aflatoxin B detection based on aptamer inherent conformation induction1Method pair AFB1High selectivity of
(1) Preparing AFB with DSMO at 1500, 2000 and 2500nM1、ZEN、OTA、FB1、MC-LR、PAT、T2The toxin solution of (a);
(2) the detection procedure of example 1 was used, in which the AFB to be detected1The solution was the solution of each toxin sample prepared in step (1), and then the fluorescence intensities at the emission wavelengths of 530nm and 582nm were recorded, and the product of the two fluorescence intensities was calculated. The results are shown in FIG. 5.
Example 5
In this embodiment, the following steps are taken to examine the signal-to-noise ratio of the detection method provided by the present invention
(1) In a buffer system (33 mM Tris-HCl (pH 7.9 at 25 ℃), 10 mM MgCl266 mM KCl) was added 3.5ul of 20x EvaGreen and 3.5ul of 10uM AFB1Diluting aptamer with ultrapure water to 31.5ul, mixing, and reacting at 25 deg.C for 5min to obtain AFB1Detecting the liquid;
the aptamer is marked with TAMRA fluorescent molecules at the 5' end, and the nucleotide sequence of the aptamer is shown as SEQ ID No.1 in a sequence table;
(2) in AFB13.5ul AFB was added to the test solution1Uniformly mixing solutions with the concentration of 2000ng/ml, incubating at 25 ℃ for 40min, determining the fluorescence intensity under the fluorescence detection condition of 488nm of excitation wavelength and 518-700nm of emission wavelength, recording the fluorescence intensity at 530nm and 582nm of emission wavelength, and calculating the product of the fluorescence intensities at the two positions;
(3) the AFB in the step (2) is treated1Replacing the solution with ultrapure water with the same amount, repeating the operations of the steps (1) and (2), recording the fluorescence intensity at the emission wavelengths of 530nm and 582nm, and calculating the product of the fluorescence intensity at the two positions;
(4) and (3) calculating the ratio of the fluorescence intensity products measured in the step (2) and the step (3) to obtain the signal-to-noise ratio, wherein the result is shown in table 1.
Comparative example 1
In this comparative example, use is made ofAFB with aptamers not labeled with TAMRA molecules1Detecting and investigating the signal-to-noise ratio
The detection principle is as follows: as shown in fig. 6, AFB1The aptamer forms a specific secondary structure through base complementary pairing self hybridization, and an organic fluorescent dye EvaGreen capable of specifically recognizing and embedding into a DNA double-stranded structure to emit light is introduced. Then introducing AFB1The original structure of the aptamer is changed due to the specific combination with the aptamer, the DNA double-chain structure is less, the fluorescence intensity of the EvaGreen is induced to be low, and finally the change of the fluorescence intensity of the EvaGreen molecule is used as a signal to output to AFB1Quantitative analysis of (3).
The experimental procedure was as follows:
(1) in a buffer system (33 mM Tris-HCl (pH 7.9 at 25 ℃), 10 mM MgCl266 mM KCl) was added 3.5ul of 20x EvaGreen and 3.5ul of 10uM AFB1Diluting aptamer with ultrapure water to 31.5ul, mixing, and reacting at 25 deg.C for 5min to obtain AFB1Detecting the liquid;
the nucleotide sequence of the aptamer is shown as SEQ ID No.1 in a sequence table;
(2) in AFB13.5ul AFB was added to the test solution1Uniformly mixing solutions with the concentration of 2000ng/ml, incubating at 25 ℃ for 40min, measuring the fluorescence intensity under the fluorescence detection condition of 488nm of excitation wavelength and 518-700nm of emission wavelength, and recording the fluorescence intensity at 530nm of emission wavelength;
(3) the AFB in the step (2) is treated1Replacing the solution with ultrapure water in the same amount, repeating the steps (1) and (2), and recording the fluorescence intensity at the emission wavelength of 530 nm;
(4) and (3) calculating the ratio of the fluorescence intensity measured in the step (2) to the fluorescence intensity measured in the step (3) to obtain the signal-to-noise ratio, wherein the result is shown in Table 1.
TABLE 1 SNR for example 5 versus comparative example 1
| Example of the implementation | Signal to noise ratio |
| Comparative example 1 | 1.39 |
| Example 5 | 6..87 |
<110> Sichuan university
120 method for detecting aflatoxin B1 based on aptamer inherent conformation induction
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence
<400> 1
gttgg gcacg tgttg tctct ctgtg tctcg tgccc ttcgc taggc ccaca 50
Claims (5)
1. Aflatoxin B detection based on aptamer inherent conformation induction1The method is characterized by comprising the following steps:
(a) preparation of Aflatoxin B1A standard curve of concentration versus fluorescence intensity;
(b) detecting the sample to be detected according to the process of preparing the standard curve to obtain the fluorescence intensity of the sample to be detected, and calculating the aflatoxin B in the sample to be detected according to the standard curve1The concentration of (c);
wherein, the step of preparing the standard curve comprises the following steps:
(1) adding fluorescent dye and AFB (fluorescent dye) for specifically recognizing double-stranded DNA (deoxyribonucleic acid) into a buffer solution system1Aptamer to prepare AFB1Detecting the liquid;
the buffer system comprises: ultrapure water, 33 mM Tris-HCl, 10 mM MgCl2, 66 mM KCl；
The pH of the Tris-HCl at 25 ℃ is 7.9;
the fluorescent dye is an EvaGreen molecule which is embedded into a DNA double-chain structure and emits light;
the aptamer is marked with TAMRA fluorescent molecules at the 5' end, and the nucleotide sequence of the aptamer is shown as SEQ ID No.1 in a sequence table;
(2) in AFB1Adding AFB with different concentrations into the detection solution1Mixing the standard solution uniformly and reacting;
(3) measuring the fluorescence intensity of the reaction mixed solution obtained in the step (2) to obtain the fluorescence intensity along with AFB1Standard curve of concentration change.
2. The method for detecting aflatoxin B of claim 11Characterized in that all reactions are carried out in a homogeneous solution in one tube.
3. The method for detecting aflatoxin B of claim 11The method is characterized in that the step (1) comprises the following steps: combining a fluorescent dye with AFB1Adding the aptamer into the buffer solution, and incubating for 0-20 min at room temperature of 15-35 ℃ to obtain AFB1And (6) detecting the liquid.
4. The method of claim 1, wherein the step (2) comprises the steps of: the prepared AFB to be tested1And (3) adding the standard solution into the solution obtained in the step (1), and reacting at 15-35 ℃ for more than 25 min.
5. The method as claimed in claim 1, wherein the fluorescence detection conditions in step (3) are an excitation wavelength of 488nm, an emission wavelength in the range of 518-700nm, and a scanning step of 1 nm.
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