CN107192816A - A kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1 - Google Patents
A kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1 Download PDFInfo
- Publication number
- CN107192816A CN107192816A CN201710339460.0A CN201710339460A CN107192816A CN 107192816 A CN107192816 A CN 107192816A CN 201710339460 A CN201710339460 A CN 201710339460A CN 107192816 A CN107192816 A CN 107192816A
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- specific probe
- microwell plate
- probe
- aptamer
- 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
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1.The method that the present invention is provided in turn includes the following steps：(1) take with coated microwell plate, add sample to be tested and specific probe is incubated altogether, be washed out；(2) by detecting that the corresponding signal of specific probe realizes the detection to aflatoxin B1；It is described that there is coated microwell plate to be the microwell plate for being coated with aflatoxin B1 BSA conjugate；The specific probe is the conjugate of element first and element second；Element first is to be connected the material that biotin is obtained as linking arm by triethylene glycol with the end of the aptamer of aflatoxin B1 specific bond；Element second is the Streptavidin of horseradish peroxidase-labeled.The enzyme-linked aptamers analysis method that the present invention is provided has advantage simple, that quick, sensitivity is high, and the quick analysis of multiple samples can be achieved, detection kit can be made, and is had great application prospect in AFB1 analysis detection.
Description
Technical field
The present invention relates to a kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1.
Background technology
Aflatoxin (Aflatoxin) is a kind of mycotoxin, is primarily referred to as what is produced by aspergillus flavus and aspergillus parasiticus
Secondary metabolite.Aflatoxin is present in a variety of contaminated food, such as corn, cereal, drinks, nut fruits, seasoning
Material and bean product.Wherein, aflatoxin B1 (Aflatoxin B1, AFB1) is a kind of toxicity most strong aflatoxin, quilt
International cancer research institution confirms as a kind of topmost carcinogenic substance.In view of AFB1 to human health and the prestige of food security
The side of body, and its contaminated popularity, the Sensitive Detection to AFB1 are very necessary.
At present, the chromatogram analysis method such as high performance liquid chromatography, liquid chromatography tandem mass spectrometry and thin-layered chromatography
It has been widely used in AFB1 quantitative analysis.Chromatogram analysis method possesses higher sensitivity and the degree of accuracy, but operation
Time-consuming, complex steps and the instrument for needing costliness.In contrast to this, immunoassay is simple to operate, quick, easily realizes scene inspection
Survey, wherein based on enzyme reaction signal amplify ELISA (Enzyme-linked immunosorbent assay,
ELISA) there is higher detection sensitivity.However, in ELISA, antibody has some limitations, such as immune antiboidy
Sensitive to environmental factor, active unstable, preparation cost height etc., and the reappearance between the experiment of different batches immune antiboidy product
It is low.
The content of the invention
It is an object of the invention to provide a kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1.
The invention provides a kind of method for detecting aflatoxin B1, in turn include the following steps：
(1) take with coated microwell plate, add sample to be tested and specific probe is incubated altogether, be washed out；
(2) by detecting that the corresponding signal of specific probe realizes the detection to aflatoxin B1；
It is described that there is coated microwell plate to be following (a) or (b) or (c)：
(a) it is coated with the microwell plate of aflatoxin B1-BSA conjugate；
(b) it is coated with the microwell plate of the conjugate of aflatoxin B1 and carrier protein；
(c) it is coated with the ELISA Plate of aflatoxin B1；
The specific probe is specific probe first or specific probe second or specific probe third；
The specific probe first is the conjugate of element first and element second；The element first is special with aflatoxin B1
The end of the aptamer of different combination connects the material that biotin is obtained by triethylene glycol as linking arm；The element second is
The Streptavidin of horseradish peroxidase-labeled；
The specific probe second is that horseradish mistake is connected in the end with the aptamer of aflatoxin B1 specific bond
The material that oxide enzyme is obtained；
The specific probe third includes affinity ligand and enzyme molecule is marked；The affinity ligand is special with aflatoxin B1
The aptamer of different combination.
In the specific probe first, the end concretely 5' ends.In the specific probe first, element first and element
Second is combined into Non-covalent binding.
In the specific probe second, the connection can be to be directly connected to or connected by linking arm.The special spy
In pin second, the connection can be to be covalently attached and can be non-covalent linking.In the specific probe second, the end is concretely
5' ends.
It is described specific as shown in the sequence 1 of sequence table with aflatoxin B1 specific bond aptamer.
The condition being incubated altogether is concretely：It is stored at room temperature incubation 30 minutes.
The microwell plate is clear microplate；The specific probe is the specific probe first or the specific probe second；
The implementation of the step (2) is：Tmb substrate solution is added, after incubation at room temperature (can specifically be stored at room temperature incubation 30 minutes)
Determine the absorbance at 450nm.
The microwell plate is nontransparent microwell plate (such as black microwell plate)；The specific probe is the specific probe first
Or the specific probe second；The implementation of the step (2) is：Chemical luminous substrate solution is added, incubation at room temperature (specifically may be used
It is stored at room temperature incubation 5 minutes) chemiluminescence intensity is determined afterwards.
The sample to be tested can be solid sample or liquid sample, such as grape wine.
The present invention also protects a kind of probe, is specific probe first or specific probe second or specific probe third.
The specific probe first is the conjugate of element first and element second；The element first is special with aflatoxin B1
The end of the aptamer of different combination connects the material that biotin is obtained by triethylene glycol as linking arm；The element second is
The Streptavidin of horseradish peroxidase-labeled；
The specific probe second is that horseradish mistake is connected in the end with the aptamer of aflatoxin B1 specific bond
The material that oxide enzyme is obtained；
The specific probe third includes affinity ligand and enzyme molecule is marked；The affinity ligand is special with aflatoxin B1
The aptamer of different combination.
In the specific probe first, the end concretely 5' ends.In the specific probe first, element first and element
Second is combined into Non-covalent binding.
In the specific probe second, the connection can be to be directly connected to or connected by linking arm.The special spy
In pin second, the connection can be to be covalently attached and can be non-covalent linking.In the specific probe second, the end is concretely
5' ends.
It is described specific as shown in the sequence 1 of sequence table with aflatoxin B1 specific bond aptamer.
The present invention also protects a kind of kit for being used to detect aflatoxin B1, including probe described in any of the above.
The kit also includes having coated microwell plate.
It is described that there is coated microwell plate to be following (a) or (b) or (c)：
(a) it is coated with the microwell plate of aflatoxin B1-BSA conjugate；
(b) it is coated with the microwell plate of the conjugate of aflatoxin B1 and carrier protein；
(c) it is coated with the ELISA Plate of aflatoxin B1.
The microwell plate is clear microplate or nontransparent microwell plate (such as black microwell plate).
The kit also includes tmb substrate solution or chemical luminous substrate solution.
The present invention also protects any kit of probe or more described in any of the above in detection aflatoxin B1
Application.
The present invention also protects probe described in any of the above preparing for detecting answering in the kit of aflatoxin B1
With.
Concretely 96 orifice plate of microwell plate described in any of the above.
Aptamer (Aptamer) be refer to the single stranded DNA that is combined with target molecule high-affinity and high specific or
RNA.In specific recognition functionally, aptamer can compare favourably with traditional antibody.Simultaneously in terms of bio-sensing, core
Sour aptamers have the advantage not available for some antibody.For example, aptamer can be entered by the chemical synthesis of low cost
Row is a large amount of to be prepared, it is easy to various functions group is introduced on aptamer, and chemical purity is high, with higher thermally-stabilised
Property, it is easy to long term storage and transport.
It is raw using the strong interaction of biotin (biotin) and Streptavidin (streptavidin) in the present invention
The aptamer of thing element mark and the Streptavidin of horseradish peroxidase-labeled are combined together, and form HRP marks
Nucleic acid aptamer probe.Streptavidin can also be replaced with Avidin (avidin).Can also be using the method being chemically bonded
Directly HRP is tagged on aptamer.
The Cleaning Principle of the present invention：AFB1 in sample to be tested and the AFB1-BSA conjugates being fixed on microwell plate are competed
Property and probe be combined, be retained on microwell plate enzyme molecule catalysis substrate reactions generation product, produce detection signal so that
Realize the detection to AFB1 (with the increase of AFB1 concentration in sample to be tested, measured signal is gradually reduced).Using extinction
During analytical photometry, using transparent microwell plate, TMB (3,3', 5,5'-tetramethylbenzidine is used
Dihydrochloride) substrate solution, carries out enzyme reaction at room temperature, then determines the absorbance at 450nm.Using chemistry hair
When light method is detected, using the microwell plate of black, using corresponding chemical luminous substrate solution, reacted, then surveyed at room temperature
Determine chemiluminescence intensity.
In the present invention, by the use of aptamer as affinity ligand Selective recognition AFB1, mark is used as using enzyme molecule
Thing is used to produce detection signal, establishes photometric analysis of extinction method and chemiluminometry based on microwell plate quantitatively to detect
AFB1 method.Using horseradish peroxidase (Horseradish peroxidase, HRP) as enzyme mark molecule, accordingly
Reaction substrate be TMB, detection method be absorption photometric detection method when, AFB1 detection be limited to 0.2nM.Using horseradish peroxidase
Enzyme (Horseradish peroxidase, HRP) is as enzyme mark molecule, using corresponding chemical luminous substrate and chemiluminescence
During detection method, detection is limited to 0.01nM.The enzyme-linked aptamers analysis method that the present invention is provided has simple, quick, sensitivity high
Advantage, can be achieved multiple samples quick analysis, detection kit can be made, AFB1 analysis detection in have it is very big
Application prospect.
Brief description of the drawings
Fig. 1 is that the aptamer marked using HRP detects AFB1 result using absorption photometry.
Fig. 2 is that the aptamer marked using HRP detects AFB1 result using chemoluminescence method.
Fig. 3 is that the aptamer marked using HRP detects AFB1 selectivity using absorption photometry.
Fig. 4 is diluted using HRP labeling nucleic acids aptamers using chemoluminescence method detection to be added in white wine sample
AFB1 result.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
Aflatoxin B1, 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, English entitled Ochratoxin A are abbreviated as OTA, the limited public affairs of Qingdao Puri nation bioengineering
Department, catalog number is STD#5012.
Fumonisin B1：The entitled fumonisin B1 of English, are abbreviated as FB1, Qingdao bioengineering Co., Ltd of Puri nation,
Catalog number is STD#2031.
Fumonisin B2：The entitled fumonisin B2 of English, are abbreviated as FB2, Qingdao bioengineering Co., Ltd of Puri nation,
Catalog number is STD#2041.
Zearalenone：The entitled zearalenone of English, is abbreviated as ZAE, the limited public affairs of Qingdao Puri nation bioengineering
Department, catalog number is STD#4012.
The Streptavidin (HRP-conjugated Streptavidin) of horseradish peroxidase-labeled：Raw work is biological
Engineering (Shanghai) limited company, production code member D111054-0100.The Streptavidin of horseradish peroxidase-labeled is again
Claim the HRP Streptavidins of mark.
AFB1-BSA conjugates：Sigma Aldriches, production code member is A6655.
Conjugate solution：AFB1-BSA conjugates are dissolved in 0.1M Na2CO3The aqueous solution (pH 9.6), AFB1-BSA couplings
The concentration of thing is 10 μ g/mL.
Association reaction cushioning liquid：10mM HEPES (pH 7.0), 20mM MgCl2, 50mM NaCl and 1mg/mLBSA.
Wash solution：10mM HEPES (pH 7.0), 20mM MgCl2, 50mM NaCl and 0.1% (volumn concentration)
Tween-20。
Lock solution：10mM HEPES (pH 7.0), 20mM MgCl2, 50mM NaCl and 10mg/mL BSA.
TMB (3,3', 5,5'-tetramethylbenzidine dihydrochloride) substrate solution：Raw work is biological
Engineering (Shanghai) limited company, production code member C006110.
Chemical luminous substrate solution：Sigma Aldriches, production code member 11582950001.In use, reagent A
(luminol and 4-iodophenol) is with reagent B (hydrogen peroxide) according to 100：1 volume ratio mixes incubation 15 minutes at room temperature.
96 transparent orifice plates：Corning companies, production code member Costar 3590.
96 orifice plates of black：Thermo Fisher Scientific companies, model NUNC Maxisorp.
Multi-function microplate reader：Synergy H1microplate reader,Biotek,USA.
Embodiment 1, the aptamer for preparing HRP marks
1st, prepare the aptamer with biotin labeling (Shanghai bioengineering Co., Ltd is artificial synthesized).
The nucleotide sequence of aptamer is as shown in the sequence 1 of sequence table, and 5 ' ends of aptamer are sweet by three
Alcohol (triethylene glycol, TEG) connects biotin as linking arm.
The sequence 1 of sequence table：5’-TGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCT-3'.
2nd, the aptamer with biotin labeling prepared by the Streptavidin and step 1 of HRP marks is according to 1：1
Mol ratio 4 DEG C of stationary incubations 30 minutes in association reaction cushioning liquid, obtain the aptamer of HRP marks.
In subsequent operation, the concentration of the aptamer of HRP marks is in terms of the DNA concentration of aptamer.
Embodiment 2, the microwell plate for preparing the modification of AFB1-BSA conjugates
First, the preparation of microwell plate first
Microwell plate first is to have the microwell plate that AFB1-BSA conjugates are modified for absorption photometric detection method.
1st, 96 transparent orifice plates are taken, 100 microlitres of conjugate solutions, 4 DEG C of stationary incubations 12 hours are added per hole.
2nd, complete after step 1, take 96 orifice plate, three times are washed with wash solution and (adds 150 microlitres of washings per hole every time molten
Liquid).
3rd, complete after step 2, take 96 orifice plate, 200 microlitres of lock solutions are added per hole, be stored at room temperature incubation 1 hour.
4th, complete after step 3, take 96 orifice plate, 300 microlitres of wash solutions are added per hole and are washed once.
Obtain microwell plate first.
2nd, the preparation of microwell plate second
Microwell plate second is to have the microwell plate that AFB1-BSA conjugates are modified for chemiluminescence detecting method.
96 transparent orifice plates are replaced with 96 orifice plates of black, other are with step one.
Obtain microwell plate second.
Embodiment 3, the aptamer marked using HRP detect AFB1 using absorption photometry
Solution to be measured：The knot of the aptamer of the HRP marks prepared containing 2nM embodiments 1 and the AFB1 of various concentrations
Close reaction cushioning liquid.The control for being added without AFB1 is set.In solution to be measured, AFB1 concentration is shown in Fig. 1 abscissa.
1st, microwell plate first prepared by Example 2,100 microlitres of solution to be measured are added per hole, are stored at room temperature incubation 30 minutes.
2nd, complete after step 1, take the microwell plate, abandon supernatant, three times are washed with wash solution and (adds 150 per hole every time micro-
Rise wash solution).
3rd, complete after step 2, take the microwell plate, 100 microlitres of tmb substrate solution are added per hole, incubation 30 is stored at room temperature
Minute, 100 microlitres of 1M HCl solutions are then added per hole.
4th, complete after step 3, take the microwell plate, the absorbance at 450nm is determined using multi-function microplate reader.
Every kind of AFB1 concentration sets 2 reprocessings, results averaged.
As a result Fig. 1 is seen.In Fig. 1, A figures show AFB1 concentration for 0,0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM,
Corresponding absorbance detection result when 20nM, 50nM, 100nM, 200nM and 500nM, B figures show AFB1 concentration for 0,
Corresponding absorbance detection result when 0.2nM, 0.5nM, 1nM and 2nM.As a result show：With the increase of AFB1 concentration, absorbance
Value is gradually reduced, and detection is limited to 0.2nM AFB1.
Embodiment 4, the aptamer marked using HRP detect AFB1 using chemoluminescence method
Solution to be measured：The knot of the aptamer of the HRP marks prepared containing 2nM embodiments 1 and the AFB1 of various concentrations
Close reaction cushioning liquid.The control for being added without AFB1 is set.In solution to be measured, AFB1 concentration is shown in Fig. 2 abscissa.
1st, microwell plate second prepared by Example 2,100 microlitres of solution to be measured are added per hole, are stored at room temperature incubation 30 minutes.
2nd, complete after step 1, take the microwell plate, abandon supernatant, three times are washed with wash solution and (adds 150 per hole every time micro-
Rise wash solution).
3rd, complete after step 2, take the microwell plate, 100 microlitres of chemical luminous substrate solution are added per hole, is stored at room temperature and incubates
Educate 5 minutes.
4th, complete after step 3, take the microwell plate, chemiluminescence intensity is determined using multi-function microplate reader.
Every kind of AFB1 concentration sets 2 reprocessings, results averaged.
As a result Fig. 2 is seen.In Fig. 2, A figures show AFB1 concentration for 0,0.01nM, 0.02nM, 0.05nM, 0.1nM,
Corresponding chemiluminescence intensity value when 0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM and 200nM, B figures
Corresponding chemiluminescence intensity when showing AFB1 concentration for 0,0.01nM, 0.02nM, 0.05nM, 0.1nM, 0.2nM and 0.5nM
Value.As a result show：With the increase of AFB1 concentration, chemiluminescence intensity value is gradually reduced, and detection is limited to 0.01nM AFB1.
Embodiment 5, the aptamer marked using HRP detect AFB1 selectivity using absorption photometry
Solution to be measured：The aptamer and the knot of different testing compounds of the HRP marks prepared containing 2nM embodiments 1
Close reaction cushioning liquid.The blank sample for being added without testing compound is set.When testing compound is aflatoxin B1, treating
The concentration surveyed in solution is 20nM.Testing compound is ochratoxin A, fumonisin B1, fumonisin B2 or Gibberella zeae
During ketenes, the concentration in solution to be measured is 100nM.
Method be the same as Example 3.
Every kind of testing compound sets 2 reprocessings, results averaged.
As a result Fig. 3 is seen.What the signal intensity that the solution to be measured only containing AFB1 is measured was measured significantly lower than blank sample
Signal intensity, and the signal intensity that the signal intensity that the solution to be measured containing each other compound is measured is measured with blank sample
It is close.As a result show, the method that the present invention is provided has good selectivity.
Embodiment 6, the aptamer marked using HRP are diluted using chemoluminescence method detection to be added in white wine sample
Plus AFB1
White wine sample (Great Wall Dry White Wine, China Great Wall Grape Wine Co., Ltd.'s production, the number of degrees 11.5%) is taken,
20 times of volumes are diluted to association reaction cushioning liquid, then add embodiment 1 prepare HRP mark aptamer and
AFB1, obtains solution to be measured.In solution to be measured, the concentration of the aptamer of HRP marks is 2nM, and AFB1 concentration is shown in Fig. 4's
Abscissa.
Method be the same as Example 4.
Every kind of AFB1 concentration sets 2 reprocessings, results averaged.
As a result Fig. 4 is seen.In Fig. 4, A figures show AFB1 concentration for 0,0.01nM, 0.02nM, 0.05nM, 0.1nM,
Corresponding chemiluminescence intensity value when 0.5nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM and 200nM, B figures are shown
Corresponding chemiluminescence intensity value when AFB1 concentration is 0,0.01nM, 0.02nM, 0.05nM, 0.1nM and 0.5nM.As a result show,
With the increase of AFB1 concentration, chemiluminescence intensity value is gradually reduced, and detection is limited to 0.01nM AFB1.
SEQUENCE LISTING
<110>Ecological Environment Research Center, Chinese Academy of Sciences
<120>A kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1
<130> GNCYX170988
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 34
<212> DNA
<213>Artificial sequence
<400> 1
tgggcacgtg ttgtctctct gtgtctcgtg ccct 34
Claims (10)
1. a kind of method for detecting aflatoxin B1, in turn includes the following steps：
(1) take with coated microwell plate, add sample to be tested and specific probe is incubated altogether, be washed out；
(2) by detecting that the corresponding signal of specific probe realizes the detection to aflatoxin B1；
It is described that there is coated microwell plate to be following (a) or (b) or (c)：
(a) it is coated with the microwell plate of aflatoxin B1-BSA conjugate；
(b) it is coated with the microwell plate of the conjugate of aflatoxin B1 and carrier protein；
(c) it is coated with the ELISA Plate of aflatoxin B1；
The specific probe is specific probe first or specific probe second or specific probe third；
The specific probe first is the conjugate of element first and element second；The element first is specifically to be tied with aflatoxin B1
The end of the aptamer of conjunction connects the material that biotin is obtained by triethylene glycol as linking arm；The element second is horseradish
The Streptavidin of peroxidase labelling；
The specific probe second is that horseradish peroxidating is connected in the end with the aptamer of aflatoxin B1 specific bond
The material that thing enzyme is obtained；
The specific probe third includes affinity ligand and enzyme molecule is marked；The affinity ligand is specifically to be tied with aflatoxin B1
The aptamer of conjunction.
2. the method as described in claim 1, it is characterised in that：The aptamer with aflatoxin B1 specific bond
As shown in the sequence 1 of sequence table.
3. method as claimed in claim 1 or 2, it is characterised in that：
The microwell plate is clear microplate；The specific probe is the specific probe first or the specific probe second；
The implementation of the step (2) is：Tmb substrate solution is added, the absorbance at 450nm is determined after incubation at room temperature.
4. method as claimed in claim 1 or 2, it is characterised in that：
The microwell plate is nontransparent microwell plate；The specific probe is the specific probe first or the specific probe second；
The implementation of the step (2) is：Chemical luminous substrate solution is added, chemiluminescence intensity is determined after incubation at room temperature.
5. a kind of probe, is specific probe first or specific probe second or specific probe third；
The specific probe first is the conjugate of element first and element second；The element first is specifically to be tied with aflatoxin B1
The end of the aptamer of conjunction connects the material that biotin is obtained by triethylene glycol as linking arm；The element second is horseradish
The Streptavidin of peroxidase labelling；
The specific probe second is that horseradish peroxidating is connected in the end with the aptamer of aflatoxin B1 specific bond
The material that thing enzyme is obtained；
The specific probe third includes affinity ligand and enzyme molecule is marked；The affinity ligand is specifically to be tied with aflatoxin B1
The aptamer of conjunction.
6. probe as claimed in claim 5, it is characterised in that：The aptamer with aflatoxin B1 specific bond
As shown in the sequence 1 of sequence table.
7. a kind of kit for being used to detect aflatoxin B1, including probe described in claim 5 or 6.
8. kit as claimed in claim 7, it is characterised in that：
The kit also includes having coated microwell plate；
It is described that there is coated microwell plate to be following (a) or (b) or (c)：
(a) it is coated with the microwell plate of aflatoxin B1-BSA conjugate；
(b) it is coated with the microwell plate of the conjugate of aflatoxin B1 and carrier protein；
(c) it is coated with the ELISA Plate of aflatoxin B1.
9. described in kit described in probe described in claim 5, probe described in claim 6, claim 7 or claim 8
Application of the kit in detection aflatoxin B1.
10. probe described in probe described in claim 5 or claim 6 is preparing the kit for detecting aflatoxin B1
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710339460.0A CN107192816B (en) | 2017-05-15 | 2017-05-15 | Method for detecting aflatoxin B1 by using enzyme-labeled aptamer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710339460.0A CN107192816B (en) | 2017-05-15 | 2017-05-15 | Method for detecting aflatoxin B1 by using enzyme-labeled aptamer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107192816A true CN107192816A (en) | 2017-09-22 |
| CN107192816B CN107192816B (en) | 2021-07-16 |
Family
ID=59872446
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710339460.0A Active CN107192816B (en) | 2017-05-15 | 2017-05-15 | Method for detecting aflatoxin B1 by using enzyme-labeled aptamer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107192816B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108709989A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A method of NeuGc ALPHA2-3Gal content is detected based on aptamer |
| CN109709321A (en) * | 2019-01-08 | 2019-05-03 | 中国科学院生态环境研究中心 | A kind of method of enzyme-linked aptamer microwell plate optical analysis small molecule |
| CN110872588A (en) * | 2019-03-04 | 2020-03-10 | 江南大学 | Aflatoxin B capable of being recognized simultaneously1、B2、G1、M1Aptamer and application thereof |
| CN111999502A (en) * | 2020-08-24 | 2020-11-27 | 湖南农业大学 | Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104374907A (en) * | 2013-09-06 | 2015-02-25 | 新疆农垦科学院 | Indirect competitive enzyme-linked adapter analysis method (ELAA) for detection of residual terramycin |
| CN105784990A (en) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for detecting aflatoxin B1 or M1 by utilizing aptamer |
-
2017
- 2017-05-15 CN CN201710339460.0A patent/CN107192816B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104374907A (en) * | 2013-09-06 | 2015-02-25 | 新疆农垦科学院 | Indirect competitive enzyme-linked adapter analysis method (ELAA) for detection of residual terramycin |
| CN105784990A (en) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for detecting aflatoxin B1 or M1 by utilizing aptamer |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108709989A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A method of NeuGc ALPHA2-3Gal content is detected based on aptamer |
| CN109709321A (en) * | 2019-01-08 | 2019-05-03 | 中国科学院生态环境研究中心 | A kind of method of enzyme-linked aptamer microwell plate optical analysis small molecule |
| CN110872588A (en) * | 2019-03-04 | 2020-03-10 | 江南大学 | Aflatoxin B capable of being recognized simultaneously1、B2、G1、M1Aptamer and application thereof |
| CN111999502A (en) * | 2020-08-24 | 2020-11-27 | 湖南农业大学 | Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth |
| CN111999502B (en) * | 2020-08-24 | 2023-08-04 | 湖南农业大学 | Aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation multimode signal output |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107192816B (en) | 2021-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singh et al. | Rapid and sensitive detection of mycotoxins by advanced and emerging analytical methods: A review | |
| CN107192816A (en) | A kind of method that utilization enzyme labeling nucleic acid aptamers detect aflatoxin B1 | |
| Reid et al. | Application of aptamers as molecular recognition elements in lateral flow assays | |
| Tang et al. | Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products | |
| Shao et al. | Biotin–streptavidin system-mediated ratiometric multiplex immunochromatographic assay for simultaneous and accurate quantification of three mycotoxins | |
| Raeisossadati et al. | Lateral flow based immunobiosensors for detection of food contaminants | |
| Turner et al. | Analytical methods for determination of mycotoxins: An update (2009–2014) | |
| Dmitrienko et al. | Recent advances in sample preparation techniques and methods of sulfonamides detection–a review | |
| Shim et al. | An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 | |
| Krska et al. | Analysis of Fusarium toxins in feed | |
| Xiong et al. | Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B1 in corn samples | |
| CN110146692A (en) | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit | |
| Majdinasab et al. | A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products | |
| Magliulo et al. | Development and validation of an ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M1 in milk | |
| Zhou et al. | Immunoassays for rapid mycotoxin detection: state of the art | |
| Zhao et al. | State of the art: Lateral flow assay (LFA) biosensor for on-site rapid detection | |
| Jeong et al. | Sensitivity and selectivity on aptamer‐based assay: The determination of tetracycline residue in bovine milk | |
| Wang et al. | Europium nanospheres-based time-resolved fluorescence for rapid and ultrasensitive determination of total aflatoxin in feed | |
| Lu et al. | Label-Free luminescent switch-on probe for ochratoxin a detection using a G-quadruplex-selective iridium (III) complex | |
| Zhu et al. | A novel fluorescence immunoassay based on AgNCs and ALP for ultrasensitive detection of sulfamethazine (SMZ) in environmental and biological samples | |
| Chen et al. | Colorimetric bio-barcode immunoassay for parathion based on amplification by using platinum nanoparticles acting as a nanozyme | |
| CN107119054A (en) | Bio-sensing probe reagent box and its application based on aptamer specific detection sulphadiazine | |
| CN108444992A (en) | A kind of quantitative aflatoxin detection kit and its detection method | |
| Wu et al. | Progress in immunoassays for nitrofurans detection | |
| Hao et al. | A chemiluminescent aptasensor for simultaneous detection of three antibiotics in milk |
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 |