CN106916822B - Method for detecting aflatoxin B1 by using aptamer molecular switch - Google Patents
Method for detecting aflatoxin B1 by using aptamer molecular switch Download PDFInfo
- Publication number
- CN106916822B CN106916822B CN201710290573.6A CN201710290573A CN106916822B CN 106916822 B CN106916822 B CN 106916822B CN 201710290573 A CN201710290573 A CN 201710290573A CN 106916822 B CN106916822 B CN 106916822B
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- aptamer
- probe
- detecting
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting aflatoxin B1 by using an aptamer molecular switch. The invention firstly protects an aptamer which is shown as a sequence 1 in a sequence table. The invention also protects a probe, which is obtained by labeling a fluorescent group FAM at the 5 'end of the single-stranded DNA molecule and labeling a quenching group BHQ1 at the 3' end. The invention also provides a method for detecting aflatoxin B1, which comprises the following steps: and co-incubating any one of the probes with a sample to be detected, wherein if the fluorescence intensity after co-incubation is reduced compared with that before co-incubation, the sample to be detected contains aflatoxin B1. The invention has the following advantages and effects: the aptamer is used as an affinity ligand for detecting the aflatoxin B1, the advantages of the aptamer can be exerted, the aptamer molecular switch can generate a response of fluorescence reduction, and the quick and sensitive detection on the aflatoxin B1 can be realized.
Description
Technical Field
The invention relates to a method for detecting aflatoxin B1 by using an aptamer molecular switch.
Background
Aflatoxins are a class of mycotoxins with strong toxicity, are produced by aspergillus flavus, aspergillus parasiticus and the like, and are important natural pollutants. Among several aflatoxins, aflatoxin B1 is the most toxic, has liver cancer-causing effect on humans and animals, and is a class 1 carcinogen. Animals and humans ingest and expose aflatoxins by eating aflatoxin contaminated foods, thereby having a significant impact on health. The method has important significance in the aspects of food safety, quality monitoring, environmental analysis and the like and has wide requirements for sensitive and rapid detection of aflatoxin.
The existing commonly used analysis method for detecting aflatoxin mainly comprises methods such as chromatography, mass spectrometry, immunoassay sensing and the like. Chromatography and mass spectrometry require the use of expensive large instruments, are costly to detect and operate and maintain, are complex to operate, and have high requirements on operators. Immune sensing analysis an immune antibody capable of specifically recognizing aflatoxin is used to construct a sensing analysis method capable of detecting aflatoxin. The preparation of immune antibody is complex and expensive, has poor stability and is easy to denature.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin B1 by using an aptamer molecular switch.
The invention firstly protects a single-stranded DNA molecule (aptamer) as shown in sequence 1 of a sequence table.
The invention also protects the application of the DNA molecule in combination with aflatoxin B1.
The invention also protects the application of the DNA molecule in detecting aflatoxin B1.
The invention also protects the application of the DNA molecule in preparing a kit for detecting aflatoxin B1.
The invention also protects a probe (probe A) which is obtained by labeling a fluorescent group at the 5 'end and labeling a quenching group at the 3' end of the single-stranded DNA molecule.
The invention also protects a probe (probe B) which is obtained by labeling a fluorescent group fluorescein FAM (fluorescein) at the 5 'end of the single-stranded DNA molecule and labeling a quenching group BHQ1(Black hole query 1) at the 3' end.
The invention also protects the application of any one of the probes in detecting aflatoxin B1.
The invention also provides a method for detecting aflatoxin B1, which comprises the following steps: and co-incubating any one of the probes with a sample to be detected, wherein if the fluorescence intensity after co-incubation is reduced compared with that before co-incubation, the sample to be detected contains aflatoxin B1.
When the probe is a probe B, the fluorescence intensity is the fluorescence intensity under the conditions that the excitation wavelength is 485 nanometers and the emission wavelength is 528 nanometers.
The co-incubation time was 15 minutes.
The co-incubation may specifically be a room temperature stationary incubation for 15 minutes.
The co-incubation is carried out in a buffer system. The pH value of the buffer system is 8.0. The buffer system comprises the following components: 10mM HEPES, 20mM MgCl2120mM NaCl, 0.1% (volume percentage content) Tween20, and the balance of water.
The probe, the sample to be detected and the buffer system form a reaction system. In the reaction system, the concentration of the probe is 25 nM.
The sample to be tested can be a solid sample or a liquid sample, such as white spirit.
The invention also protects the application of any one of the probes in preparing a kit for detecting aflatoxin B1.
The aptamer is a single-stranded nucleic acid molecule which can selectively react with a target molecule, can be prepared by a chemical synthesis method, has low synthesis cost and high stability, and is easy to introduce functional groups, such as fluorescent dye molecules and the like. The invention develops a fluorescence analysis method for detecting aflatoxin B1 by using a nucleic acid aptamer molecular switch. The 5 'end of the aptamer is labeled with a fluorescent group FAM, the 3' end of the aptamer is labeled with a quenching group BHQ1, and after the aptamer is combined with a target molecule, the fluorescence intensity of the FAM is obviously reduced, so that the detection of aflatoxin B1 is realized.
The invention has the following advantages and effects: the aptamer is used as an affinity ligand for detecting the aflatoxin B1, the advantages of the aptamer can be exerted, the aptamer molecular switch can generate a response of fluorescence reduction, and the quick and sensitive detection on the aflatoxin B1 can be realized.
Drawings
FIG. 1 shows the results of detection of aflatoxin B1 using an aptamer molecular switch.
FIG. 2 shows the results of the selectivity of the aptamer molecular switch for detecting aflatoxin B1.
Fig. 3 shows the result of detecting aflatoxin B1 added to a diluted white spirit sample using an aptamer molecular switch.
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. The aptamer was synthesized by Shanghai Bioengineering, Inc.
Buffer solution: 10mM HEPES, 20mM MgCl2120mM NaCl, 0.1% (volume percentage content) Tween20, and the balance water, pH 8.0. HEPES is known as 4-hydroxyethyl piperazine ethanesulfonic acid.
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: qingdao Pont bioengineering, Inc., catalog number STD # 5012.
Ochratoxin B: qingdao Purapang bioengineering, Inc., catalog number STD # 5021.
Fumonisins B1: qingdao Pont bioengineering, Inc., catalog number STD # 2031.
Fumonisins B2: qingdao Pont bioengineering, Inc., catalog number STD # 2041.
Zearalenone: qingdao Pont bioengineering, Inc., catalog number STD # 4012.
Example 1 preparation of specific probes
1. Artificially synthesizing a single-stranded DNA molecule (aptamer) shown in a sequence 1 in a sequence table.
2. And (3) labeling a fluorescent group FAM at the 5 'end and a quenching group BHQ1 at the 3' end of the aptamer prepared in the step 1 to obtain the specific probe.
Example 2 detection of Aflatoxin B1 Using aptamer molecular switch
The specific probe prepared in example 1, the buffer solution and aflatoxin B1 were mixed uniformly (the concentration of the probe in the initial system was 25nM, and the concentration of aflatoxin B1 is shown in the abscissa of FIG. 1), and then incubated at room temperature for 15 minutes, and then the change in fluorescence intensity of FAM (excitation wavelength 485 nM, emission wavelength 528 nM) was detected.
Each concentration setting was 2 replicates and the results averaged.
The results are shown in FIG. 1. With the increase of the concentration of aflatoxin B1, the fluorescence intensity gradually decreases, and the detection limit is 3.9 nM.
Example 3 Selective examination of Aflatoxin B1 detection Using aptamer molecular switch
The specific probe prepared in example 1, the buffer solution and the sample to be detected were mixed uniformly (in the initial system, the concentration of the probe was 25nM, and a blank control without the sample to be detected was set), and then incubated at room temperature for 15 minutes, and then the change in fluorescence intensity of FAM (excitation wavelength 485 nM, emission wavelength 528 nM) was detected.
a group: blank control.
b group: the sample to be detected is aflatoxin B1, and the concentration of aflatoxin B1 in the initial system is 200 nM.
And c, group: the sample to be detected is ochratoxin A, and the concentration of the ochratoxin A in the initial system is 1 mu M.
And d, group: the sample to be detected is ochratoxin B, and the concentration of the ochratoxin B in the initial system is 1 mu M.
And e, group: the sample to be detected is fumonisin B1, and the concentration of fumonisin B1 in the initial system is 1 mu M.
f, group: the sample to be detected is fumonisin B2, and the concentration of fumonisin B2 in the initial system is 1 mu M.
And g, group: the sample to be detected is zearalenone, and the concentration of the zearalenone in the initial system is 1 mu M.
Each set was set to 2 replicates and the results averaged.
The results are shown in FIG. 2. In fig. 2, a to g correspond to a group a to g in this order. When the sample to be detected is aflatoxin B1, the fluorescence intensity is obviously reduced compared with that of a blank control. When the sample to be detected is other substances, the fluorescence intensity has no obvious change compared with the blank control.
Example 4 detection of Aflatoxin B1 added to diluted Baijiu sample Using aptamer molecular switch
Taking a white spirit sample (Niubangshan Erguotou fen-flavor white spirit of Beijing Shuxin agriculture GmbH, the degree of which is 46%), diluting the white spirit sample to 10 times of volume by using a buffer solution, adding the specific probe prepared in example 1 and aflatoxin B1, uniformly mixing (in an initial system, the concentration of the probe is 25nM, and the concentration of aflatoxin B1 is shown in a horizontal coordinate of figure 3), standing and incubating the mixture at room temperature for 15 minutes, and detecting the change of fluorescence intensity of FAM (the excitation wavelength is 485 nM, and the emission wavelength is 528 nM).
Each concentration setting was 2 replicates and the results averaged.
The results are shown in FIG. 3. With the increase of the concentration of the aflatoxin B1, the fluorescence intensity gradually decreases, and the detection limit is 7.8 nM.
SEQUENCELISTING
<110> ecological environment research center of Chinese academy of sciences
<120> method for detecting aflatoxin B1 by using aptamer molecular switch
<130>GNCYX170936
<160>1
<170>PatentIn version 3.5
<210>1
<211>22
<212>DNA
<213> Artificial sequence
<400>1
cgtgttgtct ctctgtgtct cg 22
Claims (4)
1. The application of a probe in detecting aflatoxin B1;
the probe is obtained by labeling a fluorescent group at the 5 'end and a quenching group at the 3' end of a single-stranded DNA molecule shown in a sequence 1 of a sequence table.
2. The application of a probe in detecting aflatoxin B1;
the probe is obtained by labeling a fluorescent group FAM at the 5 'end and a quenching group BHQ1 at the 3' end of a single-stranded DNA molecule shown in a sequence 1 of a sequence table.
3. A method for detecting aflatoxin B1, comprising the following steps: co-incubating the probe of claim 1 or 2 with a test sample, wherein a decrease in fluorescence intensity after co-incubation compared to before co-incubation indicates that the test sample contains aflatoxin B1.
4. The method of claim 3, wherein: the probe is the probe as claimed in claim 2; the fluorescence intensity is the fluorescence intensity under the conditions that the excitation wavelength is 485 nanometers and the emission wavelength is 528 nanometers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710290573.6A CN106916822B (en) | 2017-04-28 | 2017-04-28 | Method for detecting aflatoxin B1 by using aptamer molecular switch |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710290573.6A CN106916822B (en) | 2017-04-28 | 2017-04-28 | Method for detecting aflatoxin B1 by using aptamer molecular switch |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106916822A CN106916822A (en) | 2017-07-04 |
| CN106916822B true CN106916822B (en) | 2020-06-19 |
Family
ID=59568696
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710290573.6A Active CN106916822B (en) | 2017-04-28 | 2017-04-28 | Method for detecting aflatoxin B1 by using aptamer molecular switch |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106916822B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318692A (en) * | 2018-01-15 | 2018-07-24 | 吉林大学 | Kanamycins is detected based on aptamer configuration switches |
| CN109490264B (en) * | 2018-11-02 | 2020-06-23 | 四川大学 | Double-end complementary aptamer probe based on aggregation luminescence and aflatoxin B1 homogeneous phase label-free detection method |
| CN110095442B (en) * | 2019-04-24 | 2020-03-03 | 中国科学院生态环境研究中心 | Method for analyzing aflatoxin B1 through fluorescence anisotropy of sensitive aptamer |
| CN110161240B (en) * | 2019-05-29 | 2020-10-09 | 福州大学 | Pseudomonas aeruginosa detection method based on aptamer fluorescence sensing |
| CN112763472B (en) * | 2020-12-29 | 2022-03-08 | 南京师范大学 | Detection system for detecting T-2 toxin residue and preparation method and application thereof |
| CN114807147B (en) * | 2021-01-19 | 2023-08-15 | 中国科学院苏州纳米技术与纳米仿生研究所 | Nucleic acid aptamer of aflatoxin B1 and application thereof |
| CN112816682B (en) * | 2021-01-28 | 2024-03-26 | 浙江省农业科学院 | Triple helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection |
| CN113866405A (en) * | 2021-10-15 | 2021-12-31 | 河南工业大学 | Preparation method of fluorescent aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102735669A (en) * | 2012-07-02 | 2012-10-17 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor |
| CN105675565A (en) * | 2016-01-24 | 2016-06-15 | 湖南科技大学 | Method for rapidly detecting aflatoxin B1 |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120225494A1 (en) * | 2009-08-21 | 2012-09-06 | Linda Chryseis Le | Dna ligands for aflatoxin and zearalenone |
-
2017
- 2017-04-28 CN CN201710290573.6A patent/CN106916822B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102735669A (en) * | 2012-07-02 | 2012-10-17 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor |
| CN105675565A (en) * | 2016-01-24 | 2016-06-15 | 湖南科技大学 | Method for rapidly detecting aflatoxin B1 |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 《Aptamer utility in sensor platforms for the detection of toxins and heavy metals》;Olasunkanml O.Olaoye等;《Journal of toxins》;20170331;第4卷(第1期);第1-12页 * |
| 《Functionalized aptamers as nano-bioprobes for ultrasensitive detection of bisphenol-A》;Ragavan K. V等;《CHEMICAL COMMUNICATIONS》;20131231;第49卷(第53期);第5960-5962页 * |
| 《基于适配体的食品中黄曲霉毒素B1和M1的生物传感器检测方法研究》;陈璐;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20160615(第6期);第18-19页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106916822A (en) | 2017-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106916822B (en) | Method for detecting aflatoxin B1 by using aptamer molecular switch | |
| CN110261362B (en) | Biosensor for simultaneously detecting zearalenone and ochratoxin A as well as preparation method and detection method thereof | |
| Hun et al. | Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A | |
| CN103852460B (en) | Based on the method that how residual the magnetic nano fluorescent sensor detection of antibiotics of aptamers is | |
| CN109406475A (en) | Double labelling quick response nucleic acid aptamer probe and its method for detecting aflatoxin B1 | |
| CN103674935B (en) | A kind of method of measuring gibberellin based on hybridization chain reaction signal amplification technique | |
| CN107129989B (en) | Aptamer for detecting aflatoxin, kit and detection method thereof | |
| CN109444101B (en) | Proportional aptamer fluorescent probe and method for detecting ochratoxin A by using same | |
| CN107084961B (en) | Aptamer molecule pair for detecting aflatoxin B1, kit and detection method thereof | |
| Lippolis et al. | Fluorescence polarisation immunoassays for rapid, accurate and sensitive determination of mycotoxins | |
| Alfonso et al. | Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure | |
| Liu et al. | Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer | |
| CN106383110B (en) | OTA chemical luminescence detection method based on nano gold mark aptamer sensor | |
| Tan et al. | Direct detection of Δ9-tetrahydrocannabinol in aqueous samples using a homogeneous increasing fluorescence immunoassay (HiFi) | |
| CN110850091A (en) | Fluorescent probe and reagent set for detecting ochratoxin A | |
| Guo et al. | Detection of aflatoxin B 1 with a new label-free fluorescent aptasensor based on exonuclease I and SYBR Gold | |
| CN106290320B (en) | A kind of OTA chemical luminescence detection method based on unmarked aptamer sensor | |
| CN108645826A (en) | A kind of new method of quick detection ascorbic acid | |
| CN113624727A (en) | Method for detecting hydrazine concentration | |
| CN108362882A (en) | A kind of fluorescence polarization immunoassay method for detecting ochratoxin | |
| Zeußel et al. | Colorimetric Method for Instant Detection of Lysine and Arginine Using Novel Meldrum's Acid‐Furfural Conjugate | |
| Dou et al. | An indirect competitive fluorescence assay for ochratoxin A based on molecular beacon | |
| CN109946274B (en) | Method for detecting aflatoxin B1 based on aptamer inherent conformation induction | |
| Huang et al. | High‐Throughput Screening for Inhibitors of the E3 Ubiquitin Ligase APC | |
| CN113913432B (en) | Tetracycline bivalent aptamer sequence and non-enzyme label-free detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |