CN110095442A - A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1 - Google Patents
A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1 Download PDFInfo
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- CN110095442A CN110095442A CN201910333449.2A CN201910333449A CN110095442A CN 110095442 A CN110095442 A CN 110095442A CN 201910333449 A CN201910333449 A CN 201910333449A CN 110095442 A CN110095442 A CN 110095442A
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- C—CHEMISTRY; METALLURGY
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of methods of sensitive aptamers fluorescence anisotropy assay aflatoxin B1.The method that the present invention protects is as follows: the aptamer of fluorochrome label, the single strand dna of marked by streptavidin and sample to be tested being reacted jointly, the fluorescence anisotropy value by measuring reaction system realizes the detection to aflatoxin B1 in sample to be tested；The aptamer can specifically bind aflatoxin B1；The single strand dna can form duplex structure with the aptamer reverse complemental；After aptamer and single strand dna reverse complemental form duplex structure, so that fluorescent dye and Streptavidin is located at the same end of duplex structure, be spatially near.The present invention has many advantages, such as sensitive, simple, quick, favorable reproducibility, is easy to high throughput analysis, amount of samples less, the easy preparation of the material used, synthesis cost is low, stability is good, storage and convenient transportation, shelf life are long, and method has high sensitivity and good selectivity.
Description
Technical field
The present invention relates to a kind of methods of sensitive aptamers fluorescence anisotropy assay aflatoxin B1.
Background technique
Aflatoxin B1 (aflatoxin B1, AFB1) is highly toxic by one kind of aspergillus flavus and aspergillus parasiticus generation
Mycotoxin, cereal, feed, fruit, dry fruit etc. are easy to be polluted by aflatoxin B1, after animal and people take in AFB1, to people
Class and the health of animal cause very big threat.AFB1 meeting induced mutation inhibits be immunized, lead to the diseases such as cancer.AFB1 is by the world
The Agency for Research on Cancer of health organization is classified as 1 grade of carcinogenic substance.Content of many countries, the world all to aflatoxin B1 in food
Propose stringent limit standard.Sensitive quick detection AFB1 is in Food Safety Analysis, environmental analysis, quality control, inlet and outlet
The fields such as trade have extensive demand.The method of common detection aflatoxin B1 mainly includes chromatography, chromatography-mass spectroscopy
Combination, mass spectrography and immunoassay sensing etc..Chromatography, mass spectrography generally require expensive instrument and equipment, and cumbersome
It is time-consuming.Immunoassay needs to identify AFB1 using immune antiboidy etc., and immunoassay sensing is comparatively more quick, but antibody
Preparation cost is high, and the stability of antibody is also bad, is easy inactivation.
Aptamer (Aptamer) be from oligonucleotide library screening obtain can be with target molecule high-affinity
The single stranded DNA or RNA combined with high specific.In specific recognition function, aptamer can be with traditional antibody phase
It matches in excellence or beauty.Simultaneously in terms of bio-sensing, aptamer has advantage not available for some antibody.For example, aptamer
It can largely be prepared by the chemical synthesis of low cost, it is easy to various functional groups are introduced on aptamer.It
Thermal stability with higher is convenient for long term storage and transport.
Summary of the invention
The object of the present invention is to provide a kind of sensitive aptamers fluorescence anisotropies (fluorescence polarization) to analyze aflatoxin
The method of B1.
This method is designed based on following principle: the aptamer of fluorochrome label and marked by streptavidin it is single-stranded
When DNA molecular hybridizes, aptamer and single strand dna reverse complemental form double-strand, and Streptavidin is close to fluorescent dye
Molecule, since the partial rotational of molecular volume increase and fluorescent dye is limited, the aptamers of fluorochrome label generate higher
Fluorescence anisotropy (fluorescence polarization) value.When aflatoxin B1, the aptamers of fluorochrome label in conjunction with target molecule,
No longer hybridize with the single strand dna of marked by streptavidin, lower fluorescence anisotropy (fluorescence is presented in detection architecture at this time
Polarization) signal.According to the reduction of signal, the detection to aflatoxin B1 may be implemented.
Present invention firstly provides a kind of methods for detecting aflatoxin B1, include the following steps: fluorescent dye mark
The aptamer of note, the single strand dna of marked by streptavidin and sample to be tested react jointly, by measuring reaction system
Fluorescence anisotropy (fluorescence polarization) value realize detection to aflatoxin B1 in sample to be tested；
The aptamer can specifically bind aflatoxin B1；
The single strand dna can form duplex structure with the partial sequence reverse complemental of the aptamer；When
After forming duplex structure, fluorescent dye and Streptavidin are located at the same end of duplex structure, and the two is spatially neighbouring.
In the method, described " fluorescent dye and Streptavidin are located at the same end of duplex structure, and the two is spatially
It is neighbouring " realized by mode shown in following (a1) or (a2):
(a1) when the partial sequence reverse complemental from 5 ' ends of single strand dna and aptamer, the fluorescence
Dye marker is in 5 ' ends of aptamer, and the marked by streptavidin is in 3 ' ends of single strand dna；
(a2) when the partial sequence reverse complemental from 3 ' ends of single strand dna and aptamer, the fluorescence
Dye marker is in 3 ' ends of aptamer, and the marked by streptavidin is in 5 ' ends of single strand dna.
In the embodiment of the present invention, single strand dna (specific as the sequence 2 to 7 of sequence table is any shown) is suitable with nucleic acid
Ligand (specifically as shown in the sequence 1 of sequence table) partial sequence reverse complemental from 5 ' ends, the fluorochrome label is in core
5 ' ends of sour aptamers, the marked by streptavidin is in 3 ' ends of single strand dna；The length of the single strand dna is
11-16bp, optimal is 14bp.
In the method, the fluorescent dye concretely fluorescein FAM.
When the fluorescent dye is fluorescein FAM, the fluorescence anisotropy value is " excitation wavelength 485nm, transmitted wave
Fluorescence anisotropy value under long 528nm ".
In the method, the aptamer, the single strand dna and sample to be tested are added jointly to reaction buffering
It is reacted in liquid.Contain 10mM Tris (pH 7.5) in the reaction buffer, 10mM MgCl2, 50mM NaCl and
0.1% (volumn concentration) Tween 20.
In the method, the concentration of the aptamer in the reaction system concretely 1nM, single strand dna
Concentration concretely 20nM.
In the method, the reaction condition concretely 4 DEG C of reaction 60min.
In the method, " detection to aflatoxin B1 in sample to be tested " is quantitative detection or qualitative detection；
When carrying out quantitative detection, according to the content for determining aflatoxin B1 in the solution to be measured as follows: when described
After reaction, the fluorescence anisotropy value measured is substituted into calibration curve equation, to calculate yellow in the sample to be tested
The content of aspertoxin B1；The calibration curve equation is obtained as follows: with the aflatoxin B1 mark of serial known concentration
Quasi- product solution is detected, and the corresponding fluorescence anisotropy value of aflatoxin B1 standard solution of each concentration is measured, thus
Obtain the calibration curve equation between the concentration and fluorescence anisotropy value of aflatoxin B1；
When carrying out qualitative detection, according to determining in the sample to be tested whether contain aflatoxin B1 as follows: working as institute
It states after reaction, if decreasing value of the measured fluorescence anisotropy value compared with blank sample fluorescence anisotropy value is greater than
3 times of blank sample signal value deviation then contain aflatoxin B1 in the sample to be tested；Conversely, the then sample to be tested
In do not contain aflatoxin B1；The blank sample signal value is carries out detection institute with without containing the solution of aflatoxin B1
The fluorescence anisotropy value measured.
The present invention also provides a kind of combinations of DNA molecular, by the aptamer and Streptavidin of fluorochrome label
The single strand dna of label forms；The aptamer can specifically bind aflatoxin B1；The single stranded DNA point
Son can form duplex structure with the partial sequence reverse complemental of the aptamer；After forming duplex structure, fluorescence dye
Material and Streptavidin are located at the same end of duplex structure, and the two is spatially neighbouring.
In DNA molecular combination, described " fluorescent dye and Streptavidin are located at the same end of duplex structure, the two
It is spatially neighbouring " realized by mode shown in following (a1) or (a2):
(a1) when the partial sequence reverse complemental from 5 ' ends of single strand dna and aptamer, the fluorescence
Dye marker is in 5 ' ends of aptamer, and the marked by streptavidin is in 3 ' ends of single strand dna；
(a2) when the partial sequence reverse complemental from 3 ' ends of single strand dna and aptamer, the fluorescence
Dye marker is in 3 ' ends of aptamer, and the marked by streptavidin is in 5 ' ends of single strand dna.
In the embodiment of the present invention, single strand dna (specific as the sequence 2 to 7 of sequence table is any shown) is suitable with nucleic acid
Ligand (specifically as shown in the sequence 1 of sequence table) partial sequence reverse complemental from 5 ' ends, the fluorochrome label is in nucleic acid
5 ' ends of aptamers, the marked by streptavidin is in 3 ' ends of single strand dna；The length of the single strand dna is 11-
16bp, optimal is 14bp.
In DNA molecular combination, the fluorescent dye concretely fluorescein FAM.
Any description above aptamer concretely single strand dna shown in the sequence 1 of sequence table.
Any description above single strand dna is concretely any one of following (b1)-(b6):
(b1) single strand dna shown in the sequence 4 of sequence table；
(b2) single strand dna shown in the sequence 2 of sequence table；
(b3) single strand dna shown in the sequence 3 of sequence table；
(b4) single strand dna shown in the sequence 5 of sequence table；
(b5) single strand dna shown in the sequence 6 of sequence table；
(b6) single strand dna shown in the sequence 7 of sequence table.
The present invention also provides the applications of any description above DNA molecular combination, for as follows (c1) or (c2):
(c1) aflatoxin B1 is detected；
(c2) kit for detecting aflatoxin B1 is prepared.
The present invention also provides the kits combined containing any description above DNA molecular；The purposes of the kit is
Detect aflatoxin B1.
It further include above-described reaction buffer in the kit.
Any description above method or DNA molecular combination or kit can be used for aspergillus flavus poison in diluted beer sample
The detection of plain B1.
The present invention detects aflatoxin B1 using fluorescence anisotropy (fluorescence polarization) analytical technology, has sensitive, simple
Single, quick, favorable reproducibility is easy to the advantages that high throughput analysis, amount of samples are few, it is only necessary to which sample and related reagent are mixed temperature
Educate measurement just.The present invention is single strand dna using aptamer and complementary nucleic acid, is easy preparation, is readily incorporated mark
Note, synthesis cost is low, stability is good, storage and convenient transportation, shelf life are long.By marked by streptavidin to mutually in this method
Mend nucleic acid end, in the detection architecture established, aptamers hybridize in the double-strand to be formed with complementary nucleic acid Streptavidin with it is glimmering
It is close on photoinitiator dye molecule space, reduce the rotation of luminescent dye molecule, this layout strategy substantially increases target molecule
In the absence of system fluorescence anisotropy value, in the presence of target molecule, it is brighter that the fluorescence anisotropy of generation reduces variation
It is aobvious, therefore detection method has high sensitivity.Under the experiment condition of optimization, method detection aspergillus flavus poison established by the present invention
Plain B1 (AFB1) detection limit reaches 60pM.Correlation technique has good selectivity.
Detailed description of the invention
Fig. 1 is the testing result in embodiment 2.
Fig. 2 is the testing result in embodiment 3.
Fig. 3 is the testing result in embodiment 4.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with and repeats to test twice, as a result makes even
Mean value.
Aflatoxin B1, the entitled Aflatoxin B1 of English, is abbreviated as AFB1, molecular formula C17H12O6, No. CAS is
1162-65-8, Qingdao bioengineering Co., Ltd, Puri nation.
Ochratoxin A, the entitled Ochratoxin A of English, is abbreviated as OTA, the limited public affairs of Qingdao Puri nation bioengineering
Department.
Fumonisin B1: the entitled fumonisin B1 of English is abbreviated as FB1, Qingdao bioengineering Co., Ltd, Puri nation.
Fumonisin B2: the entitled fumonisin B2 of English is abbreviated as FB2, Qingdao bioengineering Co., Ltd, Puri nation.
Zearalenone: the entitled zearalenone of English is abbreviated as ZAE, the limited public affairs of Qingdao Puri nation bioengineering
Department.
Streptavidin: the entitled streptavidin of English, Sangon Biotech (Shanghai) Co., Ltd., article No.:
C600432。
React buffer solution: 10mM Tris (pH 7.5), 10mM MgCl2, 50mM NaCl, 0.1% (volume basis contains
Amount) Tween 20.
DNA sequence dna used is synthetically prepared purifying by Sangon Biotech (Shanghai) Co., Ltd..
The preparation of embodiment 1, aptamer and complementary nucleic acid sequences
One, the preparation of aptamer
The artificial synthesized aptamer (as shown in the sequence 1 of sequence table) that can specifically bind aflatoxin B1,
And 5 ' ends of the aptamer are marked using fluorescein (FAM).
Two, the screening preparation of complementary nucleic acid sequences
1, for the aptamer of step 1 preparation, following several of design complementary with the aptamer can be combined
Single strand dna:
The sequence 2 of I: 5'-AGA GAC AAC ACG TGC A-3'(sequence table)；
The sequence 3 of II: 5'-GAG ACA ACA CGT GCA-3'(sequence table)；
The sequence 4 of III: 5'-AGA CAA CAC GTG CA-3'(sequence table)；
The sequence 5 of IV: 5'-GAC AAC ACG TGC A-3'(sequence table)；
The sequence 6 of V: 5'-ACA ACA CGT GCA-3'(sequence table)；
The sequence 7 of VI: 5'-CAA CAC GTG CA-3'(sequence table).
2,3 ' ends of the single strand dna for preparing step 1 use biotin labeling, then by the DNA of biotin labeling
Molecule and Streptavidin 4 DEG C of incubation 30min in reacting buffer solution according to concentration ratio 1:1, obtain marked by streptavidin
Single strand dna.
3, the marked by streptavidin for preparing the aptamer of the FAM label of step 1 preparation, step 2 is single-stranded
DNA molecular, which reacts in buffer solution with aflatoxin B1 in 100 μ l, to be mixed, 4 DEG C of incubation 60min.The nucleic acid adaptation of FAM label
The concentration of body in the reaction system is 1nM, and the concentration of the single strand dna of marked by streptavidin in the reaction system is
20nM, the concentration of aflatoxin B1 in the reaction system are 200nM.Meanwhile the blank control of sample to be tested is not added in setting.
After reaction, each using multi-function microplate reader (Synergy H1Microplate reader, Biotek, USA) measurement fluorescence
Anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
By investigating, for detecting after single strand dna labelled streptavidin shown in sequence 4, fluorescence anisotropy
Signal intensity is bigger, and it is advantageous to subsequent experimental is used for after single strand dna labelled streptavidin shown in sequence 4.
Embodiment 2 detects yellow song using the single strand dna of fluorescein labeling nucleic acid aptamers and marked by streptavidin
The method of mould toxin B1 is established
1, single shown in the sequence 4 of the aptamer, marked by streptavidin that mark the FAM prepared in embodiment 1
Ssdna molecule, which reacts in buffer solution with aflatoxin B1 in 100 μ l, to be mixed, 4 DEG C of incubation 60min.The nucleic acid of FAM label is suitable
The concentration of ligand in the reaction system is 1nM, and the concentration of the single strand dna of marked by streptavidin in the reaction system is
20nM, the aflatoxin B1 setting various concentration in reaction system (2 repetitions are arranged in each concentration).Meanwhile setting does not add
Add the blank control of sample to be tested.
2, step 1 after reaction, using multi-function microplate reader (Synergy H1Microplate reader,
Biotek, USA) measurement fluorescence anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
As a result as shown in Figure 1.In Fig. 1, abscissa is the aflatoxin B1 concentration in reaction system, and ordinate is fluorescence
Anisotropy (r) signal, when decreasing value of the sample to be tested signal compared with blank sample signal value is inclined greater than blank sample signal
3 times of difference, it is believed that detect aflatoxin B1.The results show that detection range is 60pM to 125nM, lowest detection is limited to
60pM.Maximum fluorescence anisotropy signal decreasing value is 0.144.
When carrying out quantitative detection using this method, according to determining containing for aflatoxin B1 in the solution to be measured as follows
Amount: when it is described after reaction, by the fluorescence anisotropy value measured substitute into calibration curve equation, to calculate described to be measured
The content of aflatoxin B1 in sample；The calibration curve equation is obtained as follows: with the aspergillus flavus of serial known concentration
Toxin B1 standard solution is detected, and the corresponding fluorescence anisotropy of aflatoxin B1 standard solution of each concentration is measured
Value, to obtain the calibration curve equation between the concentration of aflatoxin B1 and fluorescence anisotropy value；
When carrying out qualitative detection using this method, according to determining in the sample to be tested whether also contain aspergillus flavus as follows
Toxin B1: when it is described after reaction, if measured fluorescence anisotropy value is compared with blank sample fluorescence anisotropy value
Decreasing value be greater than 3 times of blank sample signal value deviation, then contain aflatoxin B1 in the sample to be tested；Conversely, then
Aflatoxin B1 is not contained in the sample to be tested；The blank sample signal value is with molten without containing aflatoxin B1
Liquid carries out detecting measured fluorescence anisotropy value.
The influence of embodiment 3, marked by streptavidin for detection sensitivity
It 1, will be shown in the aptamer of the FAM that prepared in embodiment 1 label, the sequence 4 only with biotin labeling
Single strand dna (not carrying out marked by streptavidin), which reacts in buffer solution with aflatoxin B1 in 100 μ l, to be mixed, and 4 DEG C
It is incubated for 60min.The concentration of the aptamer of FAM label in the reaction system is 1nM, only with the single-stranded of biotin labeling
The concentration of DNA molecular (not carrying out marked by streptavidin) in the reaction system is 20nM, the aflatoxin in reaction system
Various concentration is arranged in B1 (2 repetitions are arranged in each concentration).Meanwhile the blank control of sample to be tested is not added in setting.
2, step 1 after reaction, using multi-function microplate reader (Synergy H1Microplate reader,
Biotek, USA) measurement fluorescence anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
As a result as shown in Figure 2.The results show that with the increase of aflatoxin B1 concentration, fluorescence anisotropy signal (r)
It is gradually reduced.Detection is limited to 0.25nM aflatoxin B1.Detection range is 0.25nM to 125nM aflatoxin B1, and institute is right
The maximum fluorescence anisotropy variation answered is 0.040.
The result shows that only with the single strand dna (not carrying out marked by streptavidin) of biotin labeling, it can also be real
Now to the detection of AFB1, but detection sensitivity is low.
3, the single stranded DNA only with biotin labeling is substituted using single strand dna shown in unmarked sequence 4
Molecule is operated according to step 1 and step 2, the sensitivity detected as the result is shown and the single stranded DNA only with biotin labeling
The sensitivity of Molecular Detection is without significant difference.
4, single strand dna shown in the sequence 4 using 5 ' end labelled streptavidins is substituted only with biotin labeling
Single strand dna, operated according to step 1 and step 2, the sensitivity detected as the result is shown with it is sensitive in embodiment 2
Degree, which is compared, significant decrease.
The above results show to be detected using the complementary nucleic acid sequences of marked by streptavidin, and sensitivity is higher；Work as core
After sour aptamers are in conjunction with single strand dna, when fluorescent dye and Streptavidin are located proximate to, it may be implemented higher sensitive
Degree, detection limit are lower.
Embodiment 4, the selectivity of detection method
Sample to be tested: aflatoxin B1, ochratoxin A, ochratoxin B, fumonisin B1, fumonisin B2 and
Zearalenone.
1, by the FAM prepared in embodiment 1 label aptamer, marked by streptavidin single strand dna with
Sample to be tested mixes in 100 μ l reaction buffer solution, 4 DEG C of incubation 60min.The aptamer of FAM label is in reaction system
In concentration be 1nM, the concentration of the single strand dna of marked by streptavidin in the reaction system is 20nM, in reaction system
Sample to be tested concentration be 200nM.Meanwhile the blank control of sample to be tested is not added in setting.
2, step 1 after reaction, using multi-function microplate reader (Synergy H1Microplate reader,
Biotek, USA) measurement fluorescence anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
As a result as shown in Figure 3.In the presence of AFB1 sample, the fluorescence anisotropy signal measured is substantially reduced, and other
In the presence of the sample investigated, the corresponding signal of sample signal corresponding with blank sample solution is close, and illustration method has very
Good selectivity.
The detection of AFB1 in diluted beer sample may be implemented in method of the invention.
Sequence table
<110>Ecological Environment Research Center, Chinese Academy of Sciences
<120>a kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
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gagacaacac gtgca 15
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Claims (10)
1. a kind of method for detecting aflatoxin B1, includes the following steps: the aptamer of fluorochrome label, strepto-
The single strand dna and sample to be tested of Avidin label react jointly, real by the fluorescence anisotropy value for measuring reaction system
Now to the detection of aflatoxin B1 in sample to be tested；
The aptamer can specifically bind aflatoxin B1；
The single strand dna can form duplex structure with the partial sequence reverse complemental of the aptamer；Work as formation
After duplex structure, fluorescent dye and Streptavidin are located at the same end of duplex structure, and the two is spatially neighbouring.
2. the method as described in claim 1, it is characterised in that: described " fluorescent dye and Streptavidin are located at duplex structure
The same end, the two is spatially neighbouring " be to be realized by mode shown in following (a1) or (a2):
(a1) when the partial sequence reverse complemental from 5 ' ends of single strand dna and aptamer, the fluorescent dye
5 ' ends of aptamer are marked on, the marked by streptavidin is in 3 ' ends of single strand dna；
(a2) when the partial sequence reverse complemental from 3 ' ends of single strand dna and aptamer, the fluorescent dye
3 ' ends of aptamer are marked on, the marked by streptavidin is in 5 ' ends of single strand dna.
3. method according to claim 1 or 2, it is characterised in that: the fluorescent dye is fluorescein FAM.
4. the method as described in claims 1 to 3 is any, it is characterised in that:
The aptamer is single strand dna shown in the sequence 1 of sequence table；
The single strand dna is any one of following (b1)-(b6):
(b1) single strand dna shown in the sequence 4 of sequence table；
(b2) single strand dna shown in the sequence 2 of sequence table；
(b3) single strand dna shown in the sequence 3 of sequence table；
(b4) single strand dna shown in the sequence 5 of sequence table；
(b5) single strand dna shown in the sequence 6 of sequence table；
(b6) single strand dna shown in the sequence 7 of sequence table.
5. a kind of DNA molecular combination, by the aptamer of fluorochrome label and the single strand dna of marked by streptavidin
Composition；The aptamer can specifically bind aflatoxin B1；The single strand dna can be suitable with the nucleic acid
The partial sequence reverse complemental of ligand forms duplex structure；After forming duplex structure, fluorescent dye and Streptavidin are located at
The same end of duplex structure, the two are spatially neighbouring.
6. DNA molecular combination as claimed in claim 5, it is characterised in that: described " fluorescent dye and Streptavidin are positioned at double
The same end of chain structure, the two are spatially neighbouring " realized by mode shown in following (a1) or (a2):
(a1) when the partial sequence reverse complemental from 5 ' ends of single strand dna and aptamer, the fluorescent dye
5 ' ends of aptamer are marked on, the marked by streptavidin is in 3 ' ends of single strand dna；
(a2) when the partial sequence reverse complemental from 3 ' ends of single strand dna and aptamer, the fluorescent dye
3 ' ends of aptamer are marked on, the marked by streptavidin is in 5 ' ends of single strand dna.
7. as DNA molecular described in claim 5 or 6 combines, it is characterised in that: the fluorescent dye is fluorescein FAM.
8. the DNA molecular as described in claim 5 to 7 is any combines, it is characterised in that:
The aptamer is single strand dna shown in the sequence 1 of sequence table；
The single strand dna is any one of following (b1)-(b6):
(b1) single strand dna shown in the sequence 4 of sequence table；
(b2) single strand dna shown in the sequence 2 of sequence table；
(b3) single strand dna shown in the sequence 3 of sequence table；
(b4) single strand dna shown in the sequence 5 of sequence table；
(b5) single strand dna shown in the sequence 6 of sequence table；
(b6) single strand dna shown in the sequence 7 of sequence table.
9. the application of any DNA molecular combination of claim 5 to 7, for as follows (c1) or (c2):
(c1) aflatoxin B1 is detected；
(c2) kit for detecting aflatoxin B1 is prepared.
10. the kit containing any DNA molecular combination of claim 5 to 7；The purposes of the kit is that detection is yellow
Aspertoxin B1.
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