CN106916822A - A kind of method of utilization adaptor molecules switch detection AFB1 - Google Patents

A kind of method of utilization adaptor molecules switch detection AFB1 Download PDF

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CN106916822A
CN106916822A CN201710290573.6A CN201710290573A CN106916822A CN 106916822 A CN106916822 A CN 106916822A CN 201710290573 A CN201710290573 A CN 201710290573A CN 106916822 A CN106916822 A CN 106916822A
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afb1
probe
sample
present
aptamer
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CN106916822B (en
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赵强
王超
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Research Center for Eco Environmental Sciences of CAS
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    • 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

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of method of utilization adaptor molecules switch detection AFB1.The present invention protects a kind of aptamer first, as shown in the sequence 1 of sequence table.The present invention also protects a kind of probe, is the 5' end mark fluorophors FAM in the single strand dna, what 3 ' end mark quenching group BHQ1 were obtained.The present invention also protects a kind of method for detecting AFB1, comprises the following steps：Probe described in any of the above and sample to be tested are incubated altogether, if be incubated the preceding fluorescence intensity compared to after being incubated altogether together and declining, illustrating to contain AFB1 in sample to be tested.The present invention has the advantage that and effect：It is used to detect AFB1 as affinity ligand using aptamer, can play the advantage of aptamers, this aptamer molecular switch can produce the response of fluorescence reduction, is capable of achieving the rapid sensitive detection to AFB1.

Description

A kind of method of utilization adaptor molecules switch detection AFB1
Technical field
The present invention relates to a kind of method of utilization adaptor molecules switch detection AFB1.
Background technology
Aflatoxin is that a class has very supervirulent mycotoxin, is produced by aspergillus flavus, aspergillus parasiticus etc., is a class Important natural pollutant.In several aflatoxin, AFB1 toxicity is most strong, has to humans and animals and causes liver cancer Effect, is 1 class carcinogenic substance.Animal and people cause the intake of aflatoxin and sudden and violent by eating the food of aflatoxin contamination Dew, and then material impact is produced to health.Sensitive quick detection aflatoxin is in food security, quality monitoring, environmental analysis Etc. aspect have great importance, have extensive demand.
Conventional detection analysis methods of Aflatoxins mainly includes chromatography, mass spectrography, immunoassay sensing etc. at present Method.Chromatogram and mass spectral analysis requirement using expensive large-scale instrument, detection and operation expense high cost, complex operation, Also there is requirement higher to operating personnel.Immune sensing analysis and utilization can specific recognition aflatoxin immune antiboidy build The sensing analytical method of detectable aflatoxin.The preparation complex and expensive of immune antiboidy, less stable, easily denaturation.
The content of the invention
It is an object of the invention to provide a kind of method of utilization adaptor molecules switch detection AFB1.
The present invention protects a kind of single strand dna (aptamer) first, as shown in the sequence 1 of sequence table.
The present invention also protects application of the DNA molecular in AFB1 is combined.
The present invention also protects application of the DNA molecular in AFB1 is detected.
The present invention also protects application of the DNA molecular in the kit for being used for detecting AFB1 is prepared.
The present invention also protects a kind of probe (probe first), is the 5' end mark fluorophors in the single strand dna, What 3 ' end mark quenching groups were obtained.
The present invention also protects a kind of probe (probe second), is the 5' end mark fluorophors in the single strand dna Fluorescein FAM (fluorescein), 3 ' end mark quenching group BHQ1 (Black hole quencher 1) are obtained.
The present invention application of probe in AFB1 is detected also described in protection any of the above.
The present invention also protects a kind of method for detecting AFB1, comprises the following steps：To be visited described in any of the above Pin is incubated altogether with sample to be tested, if be incubated the preceding fluorescence intensity compared to after being incubated altogether together and declining, illustrating to contain in sample to be tested There is AFB1.
When the probe is probe second, the fluorescence intensity is for " excitation wavelength is 485 nanometers, and launch wavelength is received for 528 Fluorescence intensity under rice ".
The time of the common incubation is 15 minutes.
The common incubation is concretely stored at room temperature incubation 15 minutes.
The common incubation is carried out in buffer system.The buffer system is that pH value is 8.0.The buffer system Composition is specific as follows：10mM HEPES、20mM MgCl2, 120mM NaCl, 0.1% (volumn concentration) Tween 20, it is remaining It is water to measure.
The probe, the sample to be tested and the buffer system anabolic reaction system.In the reaction system, the spy The concentration of pin is 25nM.
The sample to be tested can be solid sample or liquid sample, such as white wine.
Present invention probe answering in the kit for being used for detecting AFB1 is prepared also described in protection any of the above With.
Aptamer is the alternative single stranded nucleic acid molecule with target molecule effect of a class, can be by chemical synthesis Method prepare, synthesize low cost, with stability higher, be readily incorporated functional group, such as luminescent dye molecule.The present invention The fluorescence analysis method that a kind of utilization aptamer molecular switch detects AFB1 is developed.The 5' of aptamer End mark fluorophor FAM, 3 ' end mark quenching group BHQ1, after it is combined with target molecule, the fluorescence intensity of FAM is bright It is aobvious to reduce, so as to realize the detection to AFB1.
The present invention has the advantage that and effect：It is used to detect aspergillus flavus poison as affinity ligand using aptamer Plain B1, can play the advantage of aptamers, and this aptamer molecular switch can produce the response of fluorescence reduction, and it is right to be capable of achieving The rapid sensitive detection of AFB1.
Brief description of the drawings
Fig. 1 is the result that AFB1 is detected using aptamer molecular switch.
Fig. 2 is the result of the selectivity of aptamer molecular switch detection AFB1.
Fig. 3 is the knot using the AFB1 added in aptamer molecular switch detection dilution Wine Sample Really.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, unless otherwise specified, is conventional method.Test material used in following embodiments, unless otherwise specified, is certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, and as a result makes even Average.Aptamer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
Cushioning liquid：10mM HEPES、20mM MgCl2, 120mM NaCl, 0.1% (volumn concentration) Tween 20, balance of water, pH 8.0.HEPES full name are 4- HEPESs.
AFB1, English entitled Aflatoxin B1, is abbreviated as AFB1, and molecular formula is C17H12O6, No. CAS is 1162-65-8, Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#1042.
Ochratoxin A：Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#5012.
Ochratoxin B：Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#5021.
Fumonisin B1：Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#2031.
Fumonisin B2：Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#2041.
Zearalenone：Qingdao bioengineering Co., Ltd of Puri nation, catalog number is STD#4012.
The preparation of embodiment 1, specific probe
1st, the single strand dna (aptamer) shown in the sequence 1 of artificial synthesized sequence table.
The sequence 1 of sequence table：5’-CGTGTTGTCTCTCTGTGTCTCG-3’.
2nd, the 5' end mark fluorophors FAM of the aptamer prepared in step 1,3 ' end mark quenching groups BHQ1, obtains specific probe.
Embodiment 2, using aptamer molecular switch detect AFB1
Specific probe prepared by embodiment 1, cushioning liquid and AFB1 mix (in initial system, probe Concentration is 25nM, and the concentration of AFB1 is shown in the abscissa of Fig. 1), incubation 15 minutes is then stored at room temperature, then detect The fluorescence intensity change (485 nanometers of excitation wavelength, 528 nanometers of launch wavelength) of FAM.
Every kind of concentration sets 2 repetitions and processes, results averaged.
Result is shown in Fig. 1.With the increase of AFB1 concentration, fluorescence intensity is gradually reduced, and detection is limited to 3.9nM.
Embodiment 3, the selectivity investigation using aptamer molecular switch detection AFB1
Specific probe prepared by embodiment 1, cushioning liquid and sample to be tested mix (in initial system, the concentration of probe It is 25nM, setting is added without the blank of sample to be tested), incubation 15 minutes is then stored at room temperature, then detect the fluorescence of FAM Strength Changes (485 nanometers of excitation wavelength, 528 nanometers of launch wavelength).
A groups：Blank.
B groups：Sample to be tested is AFB1, and the concentration of AFB1 is 200nM in initial system.
C groups：Sample to be tested is ochratoxin A, and the concentration of ochratoxin A is 1 μM in initial system.
D groups：Sample to be tested is ochratoxin B, and the concentration of ochratoxin B is 1 μM in initial system.
E groups：Sample to be tested is fumonisin B1, and the concentration of fumonisin B1 is 1 μM in initial system.
F groups：Sample to be tested is fumonisin B2, and the concentration of fumonisin B2 is 1 μM in initial system.
G groups：Sample to be tested is zearalenone, and the concentration of zearalenone is 1 μM in initial system.
2 repetitions of every group of setting are processed, results averaged.
Result is shown in Fig. 2.In Fig. 2, a to g is corresponding in turn to a groups to g groups.When sample to be tested is AFB1, with blank Control is decreased obviously compared to fluorescence intensity.When sample to be tested is other materials, fluorescence intensity is without significantly compared with blank Change.
Embodiment 4, the AFB1 using addition in aptamer molecular switch detection dilution Wine Sample
Take Wine Sample (Beijing along prosperous agriculture limited company cattle pen mountain strong, colourless liquor distilled from sorghum fen-flavor type white spirit, the number of degrees 46%) 10 times of volumes, are diluted to cushioning liquid, the specific probe and AFB1 for being subsequently adding the preparation of embodiment 1 are mixed Even (in initial system, the concentration of probe is 25nM, and the concentration of AFB1 is shown in the abscissa of Fig. 3), is then stored at room temperature It is incubated 15 minutes, then detects the fluorescence intensity change (485 nanometers of excitation wavelength, 528 nanometers of launch wavelength) of FAM.
Every kind of concentration sets 2 repetitions and processes, results averaged.
Result is shown in Fig. 3.With the increase of AFB1 concentration, fluorescence intensity is gradually reduced, and detection is limited to 7.8nM.
SEQUENCE LISTING
<110>Ecological Environment Research Center, Chinese Academy of Sciences
<120>A kind of method of utilization adaptor molecules switch detection AFB1
<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 (10)

1. a kind of single strand dna, as shown in the sequence 1 of sequence table.
2. a kind of probe, is the 5' end mark fluorophors in single strand dna described in claim 1, and 3 ' end marks are quenched Go out what group was obtained.
3. a kind of probe, is the 5' end mark fluorophor FAM in single strand dna described in claim 1,3 ' end marks Quenching group BHQ1 is obtained.
4. application of the probe described in Claims 2 or 3 in AFB1 is detected.
5. a kind of method for detecting AFB1, comprises the following steps：By probe described in Claims 2 or 3 with treat test sample This is incubated altogether, if be incubated the preceding fluorescence intensity compared to after being incubated altogether together and declining, illustrating to contain aspergillus flavus poison in sample to be tested Plain B1.
6. method as claimed in claim 5, it is characterised in that：The probe is probe described in claim 3；The fluorescence is strong It is the fluorescence intensity under " excitation wavelength is 485 nanometers, and launch wavelength is 528 nanometers " to spend.
7. application of the probe described in Claims 2 or 3 in the kit for being used for detecting AFB1 is prepared.
8. application of the DNA molecular described in claim 1 in AFB1 is combined.
9. application of the DNA molecular described in claim 1 in AFB1 is detected.
10. application of the DNA molecular described in claim 1 in the kit for being used for detecting AFB1 is prepared.
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Cited By (8)

* Cited by examiner, † Cited by third party
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
CN109490264A (en) * 2018-11-02 2019-03-19 四川大学 Based on the homogeneous label-free detection method of the luminous both-end complementary nucleic acid aptamers probe of aggregation and aflatoxin B1
CN110095442A (en) * 2019-04-24 2019-08-06 中国科学院生态环境研究中心 A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
CN110161240A (en) * 2019-05-29 2019-08-23 福州大学 A kind of pseudomonas aeruginosa detection method based on aptamers fluorescence sense
CN112763472A (en) * 2020-12-29 2021-05-07 南京师范大学 Detection system for detecting T-2 toxin residue and preparation method and application thereof
CN112816682A (en) * 2021-01-28 2021-05-18 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
CN114807147A (en) * 2021-01-19 2022-07-29 中国科学院苏州纳米技术与纳米仿生研究所 Aptamer of aflatoxin B1 and application thereof

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Cited By (11)

* Cited by examiner, † Cited by third party
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
CN109490264A (en) * 2018-11-02 2019-03-19 四川大学 Based on the homogeneous label-free detection method of the luminous both-end complementary nucleic acid aptamers probe of aggregation and aflatoxin B1
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
CN110095442A (en) * 2019-04-24 2019-08-06 中国科学院生态环境研究中心 A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
CN110161240A (en) * 2019-05-29 2019-08-23 福州大学 A kind of pseudomonas aeruginosa detection method based on aptamers fluorescence sense
CN112763472A (en) * 2020-12-29 2021-05-07 南京师范大学 Detection system for detecting T-2 toxin residue and preparation method and application thereof
CN114807147A (en) * 2021-01-19 2022-07-29 中国科学院苏州纳米技术与纳米仿生研究所 Aptamer of aflatoxin B1 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
CN112816682A (en) * 2021-01-28 2021-05-18 浙江省农业科学院 Triple helix DNA molecular switch probe and application thereof in OTA colorimetric rapid detection
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

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