CN107192816B - Method for detecting aflatoxin B1 by using enzyme-labeled aptamer - Google Patents
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Abstract
The invention discloses a method for detecting aflatoxin B1 by using an enzyme-labeled aptamer. The method provided by the invention sequentially comprises the following steps: (1) adding a sample to be detected and a specific probe into a coated microporous plate for incubation, and then washing; (2) detecting aflatoxin B1 by detecting a signal corresponding to the specific probe; the coated microporous plate is a microporous plate coated with aflatoxin B1-BSA conjugate; the specific probe is a conjugate of an element A and an element B; the element A is a substance obtained by connecting biotin at the tail end of an aptamer specifically combined with aflatoxin B1 by using triethylene glycol as a connecting arm; element b is horseradish peroxidase-labeled streptavidin. The enzyme-linked aptamer analysis method provided by the invention has the advantages of simplicity, rapidness and high sensitivity, can realize rapid analysis of a plurality of samples, can be made into a detection kit, and has a great application prospect in the analysis and detection of AFB 1.
Description
Technical Field
The invention relates to a method for detecting aflatoxin B1 by using an enzyme-labeled aptamer.
Background
Aflatoxin (Aflatoxin) is a mycotoxin, and mainly refers to a secondary metabolite produced by aspergillus flavus and aspergillus parasiticus. Aflatoxins are found in a variety of contaminated foods such as corn, cereals, wines, nuts, sauces and soy products. Among them, Aflatoxin B1(Aflatoxin B1, AFB1) is a most toxic Aflatoxin, and is recognized as a major carcinogen by international cancer research institutes. Given the threat of AFB1 to human health and food safety, and its widespread contamination, sensitive detection of AFB1 is essential.
Currently, chromatographic methods such as high performance liquid chromatography, liquid chromatography tandem mass spectrometry, and thin layer chromatography have been widely used for quantitative analysis of AFB 1. Chromatographic methods have high sensitivity and accuracy, but are time consuming, cumbersome and require expensive instrumentation. Compared with the immunoassay method, the immunoassay method is simple and rapid to operate, and is easy to realize field detection, wherein Enzyme-linked immunosorbent assay (ELISA) based on Enzyme reaction signal amplification has higher detection sensitivity. However, in the enzyme-linked immunoassay, there are some limitations to the antibody, such as sensitivity of the immune antibody to environmental factors, unstable activity, high preparation cost, and low reproducibility among different batches of immune antibody products.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin B1 by using an enzyme-labeled aptamer.
The invention provides a method for detecting aflatoxin B1, which sequentially comprises the following steps:
(1) adding a sample to be detected and a specific probe into a coated microporous plate for incubation, and then washing;
(2) detecting aflatoxin B1 by detecting a signal corresponding to the specific probe;
the coated micro-porous plate is (a) or (b) or (c) as follows:
(a) a microporous plate coated with aflatoxin B1-BSA conjugate;
(b) a microporous plate coated with a conjugate of aflatoxin B1 and a carrier protein;
(c) an ELISA plate coated with aflatoxin B1;
the specific probe is a specific probe A or a specific probe B or a specific probe C;
the specific probe A is a conjugate of the element A and the element B; the element A is a substance obtained by connecting biotin at the tail end of an aptamer specifically combined with aflatoxin B1 by using triethylene glycol as a connecting arm; the element B is streptavidin marked by horseradish peroxidase;
the specific probe B is a substance obtained by connecting horseradish peroxidase to the tail end of an aptamer specifically combined with aflatoxin B1;
the specific probe C comprises an affinity ligand and an enzyme molecular marker; the affinity ligand is an aptamer specifically combined with aflatoxin B1.
In the specific probe A, the end may be a 5' end. In the specific probe A, the combination of the element A and the element B is non-covalent combination.
In the specific probe B, the connection can be direct connection or connection through a connecting arm. In the specific probe B, the linkage may be covalent linkage and may be non-covalent linkage. In the specific probe B, the end can be specifically a 5' end.
The nucleic acid aptamer specifically combined with the aflatoxin B1 is specifically shown as a sequence 1 in a sequence table.
The co-incubation conditions may specifically be: incubation was allowed to stand at room temperature for 30 minutes.
The micropore plate is a transparent micropore plate; the specific probe is the specific probe A or the specific probe B; the step (2) is realized in the following way: the TMB substrate solution was added, and after incubation at room temperature (specifically, incubation at room temperature for 30 minutes) the absorbance at 450nm was measured.
The microplate is a non-transparent microplate (e.g., a black microplate); the specific probe is the specific probe A or the specific probe B; the step (2) is realized in the following way: adding a chemiluminescence substrate solution, and measuring the chemiluminescence intensity after room-temperature incubation (specifically, standing incubation for 5 minutes at room temperature).
The sample to be tested may be a solid sample or a liquid sample, such as wine.
The invention also protects a probe which is a specific probe A or a specific probe B or a specific probe C.
The specific probe A is a conjugate of the element A and the element B; the element A is a substance obtained by connecting biotin at the tail end of an aptamer specifically combined with aflatoxin B1 by using triethylene glycol as a connecting arm; the element B is streptavidin marked by horseradish peroxidase;
the specific probe B is a substance obtained by connecting horseradish peroxidase to the tail end of an aptamer specifically combined with aflatoxin B1;
the specific probe C comprises an affinity ligand and an enzyme molecular marker; the affinity ligand is an aptamer specifically combined with aflatoxin B1.
In the specific probe A, the end may be a 5' end. In the specific probe A, the combination of the element A and the element B is non-covalent combination.
In the specific probe B, the connection can be direct connection or connection through a connecting arm. In the specific probe B, the linkage may be covalent linkage and may be non-covalent linkage. In the specific probe B, the end can be specifically a 5' end.
The nucleic acid aptamer specifically combined with the aflatoxin B1 is specifically shown as a sequence 1 in a sequence table.
The invention also provides a kit for detecting aflatoxin B1, which comprises any one of the probes.
The kit also includes a microplate having a coating.
The coated micro-porous plate is (a) or (b) or (c) as follows:
(a) a microporous plate coated with aflatoxin B1-BSA conjugate;
(b) a microporous plate coated with a conjugate of aflatoxin B1 and a carrier protein;
(c) an ELISA plate coated with aflatoxin B1.
The microplate is a transparent microplate or a non-transparent microplate (e.g., a black microplate).
The kit also includes a TMB substrate solution or a chemiluminescent substrate solution.
The invention also protects the application of any one of the probes or any one of the kits in detecting aflatoxin B1.
The invention also protects the application of any one of the probes in preparing a kit for detecting aflatoxin B1.
Any of the microplates described above may specifically be a 96-well plate.
An Aptamer (Aptamer) refers to a single-stranded DNA or RNA that is capable of binding to a target molecule with high affinity and specificity. The nucleic acid aptamers are comparable to conventional antibodies in specific recognition function. Meanwhile, in the aspect of biosensing, the aptamer has the advantages which are not provided by some antibodies. For example, aptamers can be prepared in large quantities by low-cost chemical synthesis, various functional groups can be easily introduced into the aptamers, the chemical purity is high, the thermal stability is high, and the long-term storage and transportation are facilitated.
In the invention, by utilizing the strong interaction of biotin (biotin) and streptavidin (streptavidin), the aptamer labeled by biotin and the streptavidin labeled by horseradish peroxidase are combined together to form the nucleic acid aptamer probe labeled by HRP. Streptavidin (avidin) may also be used instead of streptavidin. The HRP can also be directly marked on the aptamer by adopting a chemical bonding method.
The detection principle of the invention is as follows: the AFB1 in the sample to be detected and the AFB1-BSA conjugate fixed on the microporous plate are competitively combined with the probe, enzyme molecules retained on the microporous plate catalyze a substrate to react to generate a product, and a detection signal is generated, so that the detection of the AFB1 is realized (the detected signal is gradually reduced along with the increase of the concentration of the AFB1 in the sample to be detected). When the absorptiometry method is used, a transparent microplate is used, an enzyme reaction is carried out at room temperature using a TMB (3,3',5,5' -tetramethylbenzidine dihydrate) substrate solution, and then the absorbance at 450nm is measured. When the chemiluminescence method is adopted for detection, a black micropore plate is adopted, a corresponding chemiluminescence substrate solution is used for reaction at room temperature, and then the chemiluminescence intensity is measured.
In the invention, the aptamer is used as an affinity ligand to selectively recognize AFB1, enzyme molecules are used as a marker to generate a detection signal, and a method for quantitatively detecting AFB1 by an absorption spectrophotometry and a chemiluminescence analysis based on a microporous plate is established. Horseradish peroxidase (HRP) is used as an enzyme labeling molecule, a corresponding reaction substrate is TMB, and when the detection method is an absorbance detection method, the detection limit of AFB1 is 0.2 nM. Horseradish peroxidase (HRP) is used as an enzyme labeling molecule, and the detection limit is 0.01nM when a corresponding chemiluminescence substrate and a corresponding chemiluminescence detection method are used. The enzyme-linked aptamer analysis method provided by the invention has the advantages of simplicity, rapidness and high sensitivity, can realize rapid analysis of a plurality of samples, can be made into a detection kit, and has a great application prospect in the analysis and detection of AFB 1.
Drawings
FIG. 1 shows the result of detecting AFB1 by absorptiometry using HRP-labeled aptamer.
FIG. 2 shows the result of detecting AFB1 by chemiluminescence using HRP-labeled nucleic acid aptamers.
FIG. 3 shows the selectivity of detection of AFB1 by absorbance using HRP-labeled aptamers.
FIG. 4 shows the result of detecting AFB1 added to a diluted white wine sample by chemiluminescence using an HRP-labeled aptamer.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Aflatoxin B1, known as Aflatoxin B1, abbreviated as AFB1, having a molecular formula of C17H12O6CAS number 1162-65-8, Qingdao Podopon bioengineering, Inc., catalog number STD # 1042.
Ochratoxin a, known under the english name Ochratoxin a, OTA for short, celand banderou bioengineering limited, under the product catalog number STD # 5012.
Fumonisins B1: the English name is fumonisin B1, abbreviated as FB1, from Qingdao Pop bioengineering, Inc., catalog number STD # 2031.
Fumonisins B2: english name is fumonisin B2, abbreviated as FB2, Islands Pont bioengineering, Inc., catalog number STD # 2041.
Zearalenone: the English name is zearalenone, abbreviated as ZAE, the Qingdao Podopanan bioengineering Co., Ltd, and the product catalog number is STD # 4012.
Horse radish peroxidase-labeled Streptavidin (HRP-conjugated Streptavidin): biotechnology engineering (Shanghai) Ltd., product number D111054-0100. The horseradish peroxidase-labeled streptavidin is also called HRP-labeled streptavidin.
AFB1-BSA conjugate: sigma Aldrich, product number A6655.
Conjugate solution: AFB1-BSA conjugate dissolved in 0.1M Na2CO3Aqueous solution (pH 9.6), AFB1-BSA conjugate concentration of 10. mu.g/mL.
Binding reaction buffer solution: 10mM HEPES (pH 7.0), 20mM MgCl250mM NaCl and 1 mg/mLBSA.
Washing solution: 10mM HEPES (pH 7.0), 20mM MgCl250mM NaCl and 0.1% (volume percent) Tween-20.
Sealing solution: 10mM HEPES (pH 7.0), 20mM MgCl250mM NaCl and 10mg/mL BSA.
TMB (3,3',5,5' -tetramethylbenzidine dihydrate) substrate solution: biometrics (Shanghai) Ltd., product number C006110.
Chemiluminescent substrate solution: sigma Aldrich, product number 11582950001. When the reagent is used, the ratio of the reagent A (luminol and 4-iodophenonol) to the reagent B (hydrogen peroxide) is as follows 100: 1 to 15 minutes at room temperature.
Clear 96-well plate: corning Inc., product number Costar 3590.
Black 96-well plates: thermo Fisher Scientific Inc., model NUNC Maxisorp.
A multifunctional microplate reader: synergy H1microplate reader, Biotek, USA.
Example 1 preparation of HRP-labeled aptamer
1. An aptamer having a biotin label was prepared (artificially synthesized by Shanghai bioengineering Co., Ltd.).
The nucleotide sequence of the aptamer is shown in sequence 1 of the sequence table, and the 5' terminal of the aptamer is connected with biotin by using triethylene glycol (TEG) as a connecting arm.
Sequence 1 of the sequence table: 5'-TGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCT-3' are provided.
2. HRP-labeled streptavidin and the nucleic acid aptamer with biotin label prepared in step 1 were mixed according to 1: 1 in a binding reaction buffer solution at 4 ℃ for 30 minutes to obtain an HRP-labeled aptamer.
In the subsequent operation, the concentration of the HRP-labeled aptamer was measured as the DNA concentration of the aptamer.
Example 2 preparation of AFB1-BSA conjugate modified microwell plate
Preparation of microporous plate A
The microplate A is a microplate with AFB1-BSA conjugate modification for absorbance detection.
1. A clear 96-well plate was added with 100. mu.l of the conjugate solution per well, and incubated at 4 ℃ for 12 hours.
2. After completion of step 1, the 96-well plate was taken and washed three times with wash solution (150 μ l of wash solution was added to each well each time).
3. After step 2, the 96-well plate was taken, 200. mu.l of the blocking solution was added to each well, and incubated at room temperature for 1 hour.
4. After completion of step 3, the 96-well plate was washed once by adding 300. mu.l of washing solution per well.
And obtaining the microporous plate A.
Preparation of microporous plate B
The microplate b is a microplate with AFB1-BSA conjugate modifications for chemiluminescence detection.
And (5) replacing the transparent 96-well plate with a black 96-well plate, and carrying out the same steps.
And obtaining the microporous plate B.
Example 3 detection of AFB1 by absorptiometry Using HRP-labeled aptamer
Solution to be tested: binding reaction buffer solutions containing 2nM of HRP-labeled aptamer prepared in example 1 and different concentrations of AFB 1. A control was set up without AFB1 addition. The concentration of AFB1 in the test solution is shown on the abscissa of fig. 1.
1. The microplate A prepared in example 2 was added with 100. mu.l of the solution to be tested per well, and incubated at room temperature for 30 minutes.
2. After step 1 was completed, the plate was removed, the supernatant discarded, and washed three times with wash solution (150 microliters of wash solution was added to each well each time).
3. And (3) after the step 2 is finished, adding 100 microliters of TMB substrate solution into each hole of the microporous plate, standing and incubating for 30 minutes at room temperature, and then adding 100 microliters of 1M HCl solution into each hole.
4. And (3) after the step 3 is finished, taking the microporous plate, and measuring the absorbance at the position of 450nm by using a multifunctional microplate reader.
The results were averaged for 2 replicates per AFB1 concentration setting.
The results are shown in FIG. 1. In FIG. 1, panel A shows the absorbance measurements at AFB1 concentrations of 0, 0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM, 200nM and 500nM, and panel B shows the absorbance measurements at AFB1 concentrations of 0, 0.2nM, 0.5nM, 1nM and 2 nM. The results show that: with increasing concentration of AFB1, the absorbance values gradually decreased with a detection limit of 0.2nM AFB 1.
Example 4 detection of AFB1 Using HRP-labeled aptamer by chemiluminescence
Solution to be tested: binding reaction buffer solutions containing 2nM of HRP-labeled aptamer prepared in example 1 and different concentrations of AFB 1. A control was set up without AFB1 addition. The concentration of AFB1 in the test solution is shown in the abscissa of fig. 2.
1. And (3) adding 100 microliters of the solution to be detected into each hole of the microporous plate B prepared in the example 2, and standing and incubating for 30 minutes at room temperature.
2. After step 1 was completed, the plate was removed, the supernatant discarded, and washed three times with wash solution (150 microliters of wash solution was added to each well each time).
3. And (3) after the step 2 is finished, adding 100 microliters of chemiluminescent substrate solution into each hole of the microporous plate, and standing and incubating for 5 minutes at room temperature.
4. And (3) after the step 3 is finished, taking the microporous plate, and determining the chemiluminescence intensity by using a multifunctional microplate reader.
The results were averaged for 2 replicates per AFB1 concentration setting.
The results are shown in FIG. 2. In FIG. 2, panel A shows the chemiluminescence intensity values corresponding to AFB1 concentrations of 0, 0.01nM, 0.02nM, 0.05nM, 0.1nM, 0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM and 200nM, and panel B shows the chemiluminescence intensity values corresponding to AFB1 concentrations of 0, 0.01nM, 0.02nM, 0.05nM, 0.1nM, 0.2nM and 0.5 nM. The results show that: as the concentration of AFB1 increased, the chemiluminescence intensity gradually decreased, with a detection limit of 0.01nM AFB 1.
Example 5 detection of selectivity of AFB1 Using HRP-labeled aptamer by absorptiometry
Solution to be tested: binding reaction buffer solutions containing 2nM of HRP-labeled aptamer prepared in example 1 and different test compounds. A blank sample was set without the test compound added. When the compound to be tested is aflatoxin B1, the concentration of the compound to be tested in the solution to be tested is 20 nM. When the compound to be tested is ochratoxin A, fumonisin B1, fumonisin B2 or zearalenone, the concentration of the compound to be tested in the solution to be tested is 100 nM.
The procedure is as in example 3.
For each test compound, 2 replicates were set and the results averaged.
The results are shown in FIG. 3. Only the solution to be tested containing AFB1 had a signal intensity significantly lower than that of the blank sample, while the solution to be tested containing each of the other compounds had a signal intensity close to that of the blank sample. The results show that the method provided by the invention has good selectivity.
Example 6 detection of added AFB1 in diluted white wine samples by chemiluminescence using HRP-labeled aptamer
A white wine sample (Changcheng dry white wine, produced by Changcheng grape wine Co., Ltd., China, degree of which is 11.5%) was diluted to 20-fold volume with a binding reaction buffer solution, and then HRP-labeled aptamer and AFB1 prepared in example 1 were added to obtain a solution to be tested. In the test solution, the concentration of the HRP-labeled aptamer was 2nM, and the concentration of AFB1 was shown in the abscissa of FIG. 4.
The procedure is as in example 4.
The results were averaged for 2 replicates per AFB1 concentration setting.
The results are shown in FIG. 4. In FIG. 4, panel A shows the chemiluminescence intensity values corresponding to AFB1 concentrations of 0, 0.01nM, 0.02nM, 0.05nM, 0.1nM, 0.5nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM and 200nM, and panel B shows the chemiluminescence intensity values corresponding to AFB1 concentrations of 0, 0.01nM, 0.02nM, 0.05nM, 0.1nM and 0.5 nM. The results show that with increasing concentration of AFB1, the chemiluminescence intensity value gradually decreased, with a detection limit of 0.01nM AFB 1.
SEQUENCE LISTING
<110> ecological environment research center of Chinese academy of sciences
<120> a method for detecting aflatoxin B1 using an enzyme-labeled aptamer
<130> GNCYX170988
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 34
<212> DNA
<213> Artificial sequence
<400> 1
tgggcacgtg ttgtctctct gtgtctcgtg ccct 34
Claims (3)
1. A method for detecting aflatoxin B1, which sequentially comprises the following steps:
(1) adding a sample to be detected and a specific probe into a coated microporous plate, standing at room temperature, incubating for 30 minutes, and washing;
(2) detecting aflatoxin B1 by detecting a signal corresponding to the specific probe;
the coated microplate is (a) or (b):
(a) a microporous plate coated with aflatoxin B1-BSA conjugate;
(b) a microporous plate coated with a conjugate of aflatoxin B1 and a carrier protein;
the specific probe is a specific probe A; the specific probe A is a conjugate of the element A and the element B; the element A is a substance obtained by connecting biotin at the tail end of an aptamer specifically combined with aflatoxin B1 by using triethylene glycol as a connecting arm; the element B is streptavidin marked by horseradish peroxidase;
the aptamer specifically bound with the aflatoxin B1 is shown as a sequence 1 in a sequence table;
the micropore plate is a non-transparent micropore plate; the step (2) is realized in the following way: adding chemiluminescence substrate solution, standing at room temperature, incubating for 5 min, and measuring chemiluminescence intensity.
2. A kit for detecting aflatoxin B1 comprises a specific probe, a coated microporous plate and a chemiluminescent substrate solution;
the specific probe is a specific probe A; the specific probe A is a conjugate of the element A and the element B; the element A is a substance obtained by connecting biotin at the tail end of an aptamer specifically combined with aflatoxin B1 by using triethylene glycol as a connecting arm; the element B is streptavidin marked by horseradish peroxidase;
the coated microplate is (a) or (b):
(a) a microporous plate coated with aflatoxin B1-BSA conjugate;
(b) a microporous plate coated with conjugate of aflatoxin B1 and carrier protein,
the aptamer specifically bound with the aflatoxin B1 is shown as a sequence 1 in a sequence table.
3. Use of the kit of claim 2 for the detection of aflatoxin B1.
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| CN104374907A (en) * | 2013-09-06 | 2015-02-25 | 新疆农垦科学院 | Indirect competitive enzyme-linked adapter analysis method (ELAA) for detection of residual terramycin |
| CN105784990A (en) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for detecting aflatoxin B1 or M1 by utilizing aptamer |
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| CN104374907A (en) * | 2013-09-06 | 2015-02-25 | 新疆农垦科学院 | Indirect competitive enzyme-linked adapter analysis method (ELAA) for detection of residual terramycin |
| CN105784990A (en) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for detecting aflatoxin B1 or M1 by utilizing aptamer |
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