CN110117641A - A method of ochratoxin A is detected using fluorescence anisotropy technology - Google Patents

A method of ochratoxin A is detected using fluorescence anisotropy technology Download PDF

Info

Publication number
CN110117641A
CN110117641A CN201910333746.7A CN201910333746A CN110117641A CN 110117641 A CN110117641 A CN 110117641A CN 201910333746 A CN201910333746 A CN 201910333746A CN 110117641 A CN110117641 A CN 110117641A
Authority
CN
China
Prior art keywords
single strand
sequence
strand dna
aptamer
ochratoxin
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.)
Granted
Application number
CN201910333746.7A
Other languages
Chinese (zh)
Other versions
CN110117641B (en
Inventor
赵强
李亚飘
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Center for Eco Environmental Sciences of CAS
Original Assignee
Research Center for Eco Environmental Sciences of CAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Research Center for Eco Environmental Sciences of CAS filed Critical Research Center for Eco Environmental Sciences of CAS
Priority to CN201910333746.7A priority Critical patent/CN110117641B/en
Publication of CN110117641A publication Critical patent/CN110117641A/en
Application granted granted Critical
Publication of CN110117641B publication Critical patent/CN110117641B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention discloses a kind of methods using fluorescence anisotropy technology detection ochratoxin A.The present invention has chosen the aptamer that can specifically bind ochratoxin A, is used fluorochrome label；The single strand dna being complementary is devised for the aptamer, is used using marked by streptavidin；After the aptamer and single strand dna combine, fluorescent dye and Streptavidin are spatially neighbouring；The aptamer of above-mentioned preparation and single strand dna and sample to be tested are reacted jointly, the fluorescence anisotropy value by measuring reaction system realizes the detection to ochratoxin A in sample to be tested.The method that the present invention establishes 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, has high sensitivity and good selectivity.

Description

A method of ochratoxin A is detected using fluorescence anisotropy technology
Technical field
The present invention relates to a kind of methods using fluorescence anisotropy technology detection ochratoxin A.
Background technique
Ochratoxin A (ochratoxin A, be abbreviated as OTA) is by several Penicilliums such as Aspergillus ochraceus mycetogenetic time Grade metabolite, international cancer research institution are classified as 2B class carcinogenic substance.Cereal (wheat, corn etc.), grain, grape, coffee Coffee etc. is often subject to the pollution of OTA.After intake polluted the food of OTA, the health of animal and human body is caused to seriously threaten.Spirit Quick, quick detection OTA has great importance and needs in food safety, quality monitoring, environmental analysis, especially field assay etc. It asks.
The method of common detection ochratoxin A mainly includes chromatography, chromatograph-mass spectrometer coupling, mass spectrography and is immunized Analysis sensing etc..Chromatography, mass spectrography generally require expensive instrument and equipment, and cumbersome time-consuming.Immunoassay needs Ochratoxin A is identified using immune antiboidy etc., and immunoassay sensing is comparatively more quick, but the preparation cost of antibody The stability of height, 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.
Relative to other analysis methods such as mass spectrum, chromatography etc., fluorescence analysis is easy to operate, sensitive, and instrument cost is low, And detection is quick.Wherein fluorescence anisotropy (fluorescence polarization) analysis method has and is not influenced, reappeared by fluorescence intensity fluctuations The good, signal stabilization of property is easy to the advantages such as high throughput analysis.Fluorescence anisotropy and fluorescence polarization are for same physical phenomenon Different expression, the two have difference in calculation formula, and the two can be mutually converted to by appropriate formula.Fluorescence is respectively to different Property and two kinds of expression of fluorescence polarization are all more commonly used.
Summary of the invention
The object of the present invention is to provide a kind of methods using fluorescence anisotropy technology detection ochratoxin A.
The design principle of this method is as follows: 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 ochratoxin A, 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 ochratoxin A may be implemented.
Present invention firstly provides a kind of methods for detecting ochratoxin A, include the following steps: fluorochrome label Aptamer, marked by streptavidin single strand dna and sample to be tested react jointly, pass through measurement reaction system Fluorescence anisotropy (fluorescence polarization) value realizes the detection to ochratoxin A in sample to be tested；
The aptamer can specifically bind ochratoxin A；
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 9 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 9- 16bp, optimal is 13bp.
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 is 5nM, and the concentration of single strand dna is 10nM。
In the method, the reaction condition is 25 DEG C of incubation 10min.
In the method, " detection to ochratoxin A in sample to be tested " is quantitative detection or qualitative detection；
When carrying out quantitative detection, according to the content for determining Ochratoxin A in the solution to be measured as follows: when the reaction After, the fluorescence anisotropy value measured is substituted into calibration curve equation, to calculate Aspergillus ochraceus in the sample to be tested The content of plain A；The calibration curve equation is obtained as follows: with the Ochratoxin A standard solution of serial known concentration into Row detection, measures the corresponding fluorescence anisotropy value of Ochratoxin A standard solution of each concentration, to obtain Ochratoxin A Concentration and fluorescence anisotropy value between calibration curve equation；
When carrying out qualitative detection, according to determining in the sample to be tested whether contain Ochratoxin A as follows: when described anti- After answering, if decreasing value of the measured fluorescence anisotropy value compared with blank sample fluorescence anisotropy value is greater than blank 3 times of sample signal value deviation then contain Ochratoxin A in the sample to be tested；Conversely, not contained in the sample to be tested then Ochratoxin A；The blank sample signal value be with the solution for not containing Ochratoxin A detect measured fluorescence respectively to Opposite sex value.
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 DNA molecular combination, the length of the single strand dna concretely 9-16bp, optimal is 13bp.
In the embodiment of the present invention, single strand dna (specific as the sequence 2 to 9 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 9- 16bp, optimal is 13bp.
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 any one of following (b1)-(b8):
(b1) single strand dna shown in the sequence 5 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 4 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；
(b7) single strand dna shown in the sequence 8 of sequence table；
(b8) single strand dna shown in the sequence 9 of sequence table.
The present invention also provides the applications of any description above DNA molecular combination, for as follows (c1) or (c2):
(c1) ochratoxin A is detected；
(c2) kit for detecting ochratoxin A is prepared.
The present invention also provides the kits combined containing any description above DNA molecular；The purposes of the kit is Detect ochratoxin A.
It further include above-described reaction buffer in the kit.
Any description above method or DNA molecular combination or aptamer or kit can be used for diluted beer sample The detection of ochratoxin A in product.
The present invention detects ochratoxin A 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 ochraceus poison established by the present invention Plain A (OTA) detection limit reaches 1nM.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.
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.
Ochratoxin A, the entitled Ochratoxin A of English, is abbreviated as OTA, molecular formula C20H18ClNO6, No. CAS is 666236-28-8, Qingdao bioengineering Co., Ltd, Puri nation.
Aflatoxin B1, the entitled Aflatoxin B1 of English, is abbreviated as AFB1, the limited public affairs of Qingdao Puri nation bioengineering Department, article No.: MSS1003.
Ochratoxin B, the entitled ochratoxin B of English, is abbreviated as OTB, the limited public affairs of Qingdao Puri nation bioengineering Department, article No.: MSS1044.
Fumonisin B1: the entitled fumonisin B1 of English, it is abbreviated as FB1, Qingdao bioengineering Co., Ltd, Puri nation, Article No.: MSS1013.
Fumonisin B2: the entitled fumonisin B2 of English, it is abbreviated as FB2, Qingdao bioengineering Co., Ltd, Puri nation, Article No.: MSS1014.
Zearalenone: the entitled zearalenone of English is abbreviated as ZAE, the limited public affairs of Qingdao Puri nation bioengineering Department, article No.: MSS1024.
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 ochratoxin A, and And 5 ' ends of the aptamer are marked using fluorescein (FAM).
Two, the screening preparation of complementary nucleic acid sequences
For the aptamer of step 1 preparation, what following several of design complementary with the aptamer can combine Single strand dna:
I: 5'-CCA CCC ACA CCC GAT C-3 ' (sequence 2 of sequence table)；
II: 5'-CAC CCA CAC CCG ATC-3 ' (sequence 3 of sequence table)；
III: 5'-ACC CAC ACC CGA TC-3 ' (sequence 4 of sequence table)；
IV: 5'-CCC ACA CCC GAT C-3 ' (sequence 5 of sequence table)；
V: 5'-CCA CAC CCG ATC-3 ' (sequence 6 of sequence table)；
VI: 5'-CAC ACC CGA TC-3 ' (sequence 7 of sequence table)；
VII: 5'-ACA CCC GAT C-3 ' (sequence 8 of sequence table)；
VIII: 5'-CAC CCG ATC-3 ' (sequence 9 of 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 ochratoxin A in 100 μ l, to be mixed, 25 DEG C of incubation 10min.The nucleic acid adaptation of FAM label The concentration of body in the reaction system be 5nM, the concentration of the single strand dna of marked by streptavidin in the reaction system be for 10nM, the concentration of ochratoxin A in the reaction system are 200nM.Meanwhile the blank control of sample to be tested is not added in setting. After reaction, fluorescence is measured using multi-function microplate reader (Synergy H1 Microplate reader, Biotek, USA) Anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
By investigating, for detecting after single strand dna labelled streptavidin shown in sequence 5, 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 5.
Embodiment 2 detects reddish brown song using the single strand dna of fluorescein labeling nucleic acid aptamers and marked by streptavidin The method of mould toxin A is established
1, by the FAM prepared in embodiment 1 label aptamer, marked by streptavidin single strand dna with Ochratoxin A mixes in 100 μ l reaction buffer solution, 25 DEG C of incubation 10min.The aptamer of FAM label is reacting Concentration in system is 5nM, and the concentration of the single strand dna of marked by streptavidin in the reaction system is 10nM, reaction Ochratoxin A setting various concentration in system (2 repetitions are arranged in each concentration).Meanwhile sample to be tested is not added in setting Blank control.
2, step 1 after reaction, using multi-function microplate reader (Synergy H1 Microplate 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 ochratoxin A 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 ochratoxin A.The results show that detection range is 1nM to 5000nM, lowest detection is limited to 1nM, Maximum fluorescence anisotropy signal decreasing value is 0.099.
When carrying out quantitative detection using this method, according to the content for determining Ochratoxin A in the solution to be measured as follows: The fluorescence anisotropy value measured is substituted into calibration curve equation after reaction when described, to calculate described to test sample The content of Ochratoxin A in this；The calibration curve equation is obtained as follows: with the Ochratoxin A mark of serial known concentration Quasi- product solution is detected, and the corresponding fluorescence anisotropy value of Ochratoxin A standard solution of each concentration is measured, to obtain Calibration curve equation between the concentration and fluorescence anisotropy value of Ochratoxin A；
When carrying out qualitative detection using this method, according to determining in the sample to be tested whether also contain Aspergillus ochraceus as follows Plain A: when it is described after reaction, if drop of the measured fluorescence anisotropy value compared with blank sample fluorescence anisotropy value Low value is greater than 3 times of blank sample signal value deviation, then contains Ochratoxin A in the sample to be tested；Conversely, then described to be measured Ochratoxin A is not contained in sample；The blank sample signal value is surveyed to be detected with the solution without containing Ochratoxin A The fluorescence anisotropy value obtained.
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 5 only with biotin labeling Single strand dna (not carrying out marked by streptavidin), which reacts in buffer solution with ochratoxin A in 100 μ l, to be mixed, and 25 DEG C It is incubated for 10min.The concentration of the aptamer of FAM label in the reaction system is 5nM, only with the single-stranded of biotin labeling The concentration of DNA molecular (not carrying out marked by streptavidin) in the reaction system is 10nM, the ochratoxin A in reaction system 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 H1 Microplate reader, Biotek, USA) measurement fluorescence anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
The results show that only with biotin labeling single strand dna (not carrying out marked by streptavidin) when, blank sample The corresponding fluorescence signal value of product is 0.098.In the presence of 200nM ochratoxin A, corresponding fluorescence anisotropy value is 0.096, The two difference is only 0.002, compared with Example 2, signal intensity very little caused by ochratoxin A.Using Streptavidin When the single strand dna of label, in the case that other experiment conditions are all the same, the corresponding fluorescence anisotropy signal of blank sample In the presence of 0.186,200nM ochratoxin A, corresponding fluorescence anisotropy value is 0.124, fluorescence anisotropy signal drop Low 0.062, signal intensity amplitude is much higher than the signal intensity value generated when the single strand dna using biotin labeling (0.002)。
3, the single stranded DNA only with biotin labeling is substituted using single strand dna shown in unmarked sequence 5 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.
The above results show to be detected using the complementary nucleic acid sequences of marked by streptavidin, when aptamer with After single strand dna combines, when fluorescent dye and Streptavidin are located proximate to, higher sensitivity, detection limit may be implemented It is lower.
Embodiment 4, the selectivity of detection method
Sample to be tested: ochratoxin A, ochratoxin B, aflatoxin B1, 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, 25 DEG C of incubation 10min.The aptamer of FAM label is in reaction system In concentration be 5nM, the concentration of the single strand dna of marked by streptavidin in the reaction system is 10nM, 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 H1 Microplate reader, Biotek, USA) measurement fluorescence anisotropy (fluorescence polarization) value, excitation wavelength 485nm, launch wavelength 528nm.
As a result as shown in Figure 2.In the presence of ochratoxin A sample, the fluorescence anisotropy signal measured is substantially reduced, And in the presence of the sample that other are investigated, the corresponding signal of sample signal corresponding with blank sample solution is close, illustration method With good selectivity.
The detection of ochratoxin A 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 using fluorescence anisotropy technology detection ochratoxin A
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatcgggtgt gggtggcgta aagggagcat cggaca 36
<210> 2
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccacccacac ccgatc 16
<210> 3
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cacccacacc cgatc 15
<210> 4
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acccacaccc gatc 14
<210> 5
<211> 13
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cccacacccg atc 13
<210> 6
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccacacccga tc 12
<210> 7
<211> 11
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cacacccgat c 11
<210> 8
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
acacccgatc 10
<210> 9
<211> 9
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cacccgatc 9

Claims (10)

1. a kind of method for detecting ochratoxin A, 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 ochratoxin A in sample to be tested；
The aptamer can specifically bind ochratoxin A；
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)-(b8):
(b1) single strand dna shown in the sequence 5 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 4 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；
(b7) single strand dna shown in the sequence 8 of sequence table；
(b8) single strand dna shown in the sequence 9 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 ochratoxin A；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)-(b8):
(b1) single strand dna shown in the sequence 5 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 4 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；
(b7) single strand dna shown in the sequence 8 of sequence table；
(b8) single strand dna shown in the sequence 9 of sequence table.
9. the application of any DNA molecular combination of claim 5 to 7, for as follows (c1) or (c2):
(c1) ochratoxin A is detected；
(c2) kit for detecting ochratoxin A is prepared.
10. the kit containing any DNA molecular combination of claim 5 to 7；The purposes of the kit is that detection is reddish brown Aspertoxin A.
CN201910333746.7A 2019-04-24 2019-04-24 Method for detecting ochratoxin A by using fluorescence anisotropy technology Active CN110117641B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910333746.7A CN110117641B (en) 2019-04-24 2019-04-24 Method for detecting ochratoxin A by using fluorescence anisotropy technology

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910333746.7A CN110117641B (en) 2019-04-24 2019-04-24 Method for detecting ochratoxin A by using fluorescence anisotropy technology

Publications (2)

Publication Number Publication Date
CN110117641A true CN110117641A (en) 2019-08-13
CN110117641B CN110117641B (en) 2021-02-09

Family

ID=67521297

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910333746.7A Active CN110117641B (en) 2019-04-24 2019-04-24 Method for detecting ochratoxin A by using fluorescence anisotropy technology

Country Status (1)

Country Link
CN (1) CN110117641B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111751346A (en) * 2020-07-08 2020-10-09 苏州健雄职业技术学院 OTA aptamer, complementary sequence thereof and OTA fluorescence detection method

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103575713A (en) * 2013-11-01 2014-02-12 山西大学 Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103575713A (en) * 2013-11-01 2014-02-12 山西大学 Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
康丽萍: "高灵敏荧光各向异性核酸探针的构建及其应用研究", 《中国优秀硕士学位论文全文数据库》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111751346A (en) * 2020-07-08 2020-10-09 苏州健雄职业技术学院 OTA aptamer, complementary sequence thereof and OTA fluorescence detection method
CN111751346B (en) * 2020-07-08 2022-10-11 苏州健雄职业技术学院 OTA aptamer, complementary sequence thereof and OTA fluorescence detection method

Also Published As

Publication number Publication date
CN110117641B (en) 2021-02-09

Similar Documents

Publication Publication Date Title
Chen et al. A simple aptamer-based fluorescent assay for the detection of Aflatoxin B1 in infant rice cereal
Wu et al. Highly sensitive fluorescence sensing of zearalenone using a novel aptasensor based on upconverting nanoparticles
Li et al. A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize
CN107271668B (en) A kind of method and kit of multi signal detection mycotoxin
Zhang et al. A fluorescent aptasensor based on DNA-scaffolded silver nanoclusters coupling with Zn (II)-ion signal-enhancement for simultaneous detection of OTA and AFB1
Turner et al. Analytical methods for determination of mycotoxins: An update (2009–2014)
Samokhvalov et al. Use of anchor protein modules in fluorescence polarisation aptamer assay for ochratoxin A determination
Hayat et al. Recent advances in ochratoxin A-producing fungi detection based on PCR methods and ochratoxin A analysis in food matrices
Tittlemier et al. Developments in mycotoxin analysis: an update for 2019-2020
Niazi et al. Simultaneous detection of fumonisin B 1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF 4: Ce, Tb and NH 2-Eu/DPA@ SiO 2) as labels
Heyduk et al. Fluorescent homogeneous immunosensors for detecting pathogenic bacteria
Long et al. Fluorescence resonance energy transfer based aptasensor for the sensitive and selective detection of 17β-estradiol using a quantum dot-bioconjugate as a nano-bioprobe
CN106916822A (en) A kind of method of utilization adaptor molecules switch detection AFB1
Xu et al. Dual fluorescent immunochromatographic assay for simultaneous quantitative detection of citrinin and zearalenone in corn samples
CN107129989B (en) Aptamer for detecting aflatoxin, kit and detection method thereof
Xie et al. Quantitative and rapid detection of amantadine and chloramphenicol based on various quantum dots with the same excitations
Kim et al. Aptamer-based fluorescent assay for simple and sensitive detection of fipronil in liquid eggs
CN109358027A (en) A kind of construction method for the aptamers biosensor measuring ochratoxin A
Song et al. Application of the Dimeric G-Quadruplex and toehold-mediated strand displacement reaction for fluorescence biosensing of ochratoxin A
Hong et al. A universal, portable, and ultra-sensitive pipet immunoassay platform for deoxynivalenol detection based on dopamine self-polymerization-mediated bioconjugation and signal amplification
CN110850091A (en) Fluorescent probe and reagent set for detecting ochratoxin A
CN110095442A (en) A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
CN108362882A (en) A kind of fluorescence polarization immunoassay method for detecting ochratoxin
CN110117641A (en) A method of ochratoxin A is detected using fluorescence anisotropy technology
Huang et al. Multiplex bio-barcode based fluorometric immunoassay for simultaneous determination of zearalenone, fumonisin B1, ochratoxin A, and aflatoxin B1 in cereals

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