CN105505940B - Aflatoxin B1 aptamer, DNA sensor and kit and application - Google Patents
Aflatoxin B1 aptamer, DNA sensor and kit and application Download PDFInfo
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- CN105505940B CN105505940B CN201610019712.7A CN201610019712A CN105505940B CN 105505940 B CN105505940 B CN 105505940B CN 201610019712 A CN201610019712 A CN 201610019712A CN 105505940 B CN105505940 B CN 105505940B
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Abstract
The present invention provides aflatoxin B1 aptamer, DNA sensor and kit and application, which has following nucleotide sequence: 5 '-GTTmmmmmmmTGTTGTCTCTCT GTGTCTnnnnnnnTTCGCTAGGCCCACA-3 '；Wherein, each m and n is each independently A, T, C or G and m segment and n segment complementary pairing.According to the present invention, measurement aflatoxin B1 is not necessarily to large-scale instrument and equipment, at low cost.Isothermal reaction can be realized the qualitative detection of naked eyes, also can be realized quantitative detection, also, be not necessarily to fluorescent dye, greatly reduces cost.
Description
Technical field
The present invention relates to field of biological detection, more particularly, to a kind of aflatoxin B1 aptamer, including this
The DNA sensor of aptamer, including the kit of the DNA sensor and the DNA sensor and kit.
Background technique
Aflatoxin be mainly by aspergillus flavus, aspergillus parasiticus generate secondary metabolite, in damp-heat area food and
Occurs the probability highest of aflatoxin in feed.They are present in soil, animals and plants, in various nuts, are especially easy dirt
The grain oil products such as dyeing flower life, corn, rice, soybean, wheat, be mycotoxicosis it is maximum, to human health risk extremely
One kind mycotoxin outstanding.The most common with aflatoxin B1 in natural food, harmfulness is also most strong.
Current existing Determination Methods of Aflatoxins mainly has:
(1) thin layer chromatography
Thin-layer chromatography is most widely used isolation technics in terms of aflatoxin research.From nineteen ninety, it is listed in AOAC
(Association of Official Agricultural Chemists) standard method, this method have simultaneously it is qualitative and
The function of quantitative analysis aflatoxin.
(2) liquid chromatography
Liquid chromatogram and thin-layer chromatography have similitude in many aspects, and the two complements each other.Usually early period is carried out with TLC
Condition setting, select the quantitative determination for carrying out aflatoxin after suitable separation condition with LC again.
(3) immunochemistry analysis method
Utilize the immunoassay of the aflatoxin of monoclonal antibody or polyclonal antibody design with high specificity
Method is also common Determination Methods of Aflatoxins.Such methods generally include radioimmunoassay method, enzyme-linked immunization
And immunochromatographic method, they can quantitative determine aflatoxin.
But these methods are at high cost, and need the instrument using Large expensive, it is cumbersome.
Aptamer is section of DNA (DNA), RNA (ribonucleic acid) either XNA (nucleic acid analog)
Sequence.Usually utilize in-vitro screening technology --- the Fas lignand system evolution technology (Systematic of index concentration
Evolution of ligands by exponential enrichment, SELEX), obtained in nucleic acid molecule libraries
Oligonucleotide fragment.Aptamer can be therefore widely used in life with target substance high specific, with high selectivity combined
Object sensor field.
DNA sensor believes the biology acted between DNA and other substances using DNA as sensing element, by energy converter
Number it is changed into the physical signals such as detectable light, electricity, sound wave.In recent years, DNA sensor is in gene diagnosis, environmental monitoring, drug
The application study in the fields such as research is in widespread attention.
Summary of the invention
The purpose of the present invention is overcoming the drawbacks described above of the prior art, provide a kind of aflatoxin B1 aptamer and
DNA sensor and kit can be realized the detection of easy to operate, high sensitivity, aflatoxin B1 at low cost.
To achieve the goals above, the present invention provides a kind of aflatoxin B1 aptamer, aptamer tool
There is following nucleotide sequence:
5 '-GTTmmmmmmmTGTTGTCTCTCTGTGTCTnnnnnnnTTCGCTAGGCCCACA-3 ' (SEQ ID NO:
1)；Wherein, each m and n is each independently A, T, C or G and m segment and n segment complementary pairing.
The present invention also provides a kind of DNA sensor, which is made of following three parts:
(1) DNA (A): the circular DNA template being formed by connecting by following DNA sequence dna 5 ' end and 3 ' ends: 5 '-
XaACCCAACCCGCCCTACCCXb-3 ' (SEQ ID NO:2)；Wherein, Xa and Xb respectively represents a and b any bases, and a+
B=40-80；
(2) DNA (B): above-mentioned aflatoxin B1 aptamer；
(3) DNA (C): RCA primer, the length of the DNA (C) is 25-50nt and sequence has the feature that 5 ' ends
5 ' flank complementary pairings of 15-30 base and DNA (A) fixed sequence program, the 10-20 base at 3 ' ends and the 5 ' ends of DNA (B) are mutual
It recruits pair.
Wherein, DNA (A) fixed sequence program is the sequence between Xa and Xb, since DNA (A) is cyclic DNA mould
Plate, therefore, 5 ' flanks of DNA (A) fixed sequence program are not limited to a part in Xa, also may include at least one in Xb
Point.The complementary series of the fixed sequence program can be catalyzed the oxidation of ABTS in the presence of hemin.
In the present invention, the RCA refers to rolling circle amplification (rolling circle amplification, RCA), the term
Meaning and technical step be known to the skilled person.
Preferably, the DNA (A) has following nucleotide sequence:
5’-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3’
(SEQ ID NO:3).
Preferably, the DNA (C) has following nucleotide sequence:
5 '-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3 ' (SEQ ID NO:4).
Or preferably, the DNA (C) has following nucleotide sequence:
5 '-TAATATCAGTCACGGTATTCTTTTCACAACAGCACGGGAAC-3 ' (SEQ ID NO:5).
Again or preferably, the DNA (C) has following nucleotide sequence:
5 '-TAATATCAGTCACGGTATTCTTTTCAGAGACAACAGCACGGGAAC-3 ' (SEQ ID NO:6).
DNA sensor of the present invention can be obtained by mixing above-mentioned three kinds of components progress self assembly.
The present invention also provides a kind of kits, which includes following components:
(1) aflatoxin B1 extracting solution；
(2) DNA sensor solution；
(3) archaeal dna polymerase；
(4) ABTS reduction-state colorless substrate；
(5) hemin solution (hemin solution)；
(6) hydrogen peroxide；
(7) HEPES buffer solution；
Wherein, DNA sensor solution includes following components:
Above-mentioned DNA sensor, RCA reaction solution and dNTP.
Wherein, the concentration of DNA (A), DNA (B) and DNA (C) are both preferably 10-1000nM in the DNA sensor solution,
Further preferably 20-100nM.
Wherein, the aflatoxin B1 extracting solution can be any solution that can dissolve aflatoxin B1, preferably
Concentration for methanol solution, the methanol solution can according to need determination, for example, can be 70% methanol solution.
Wherein, the hydrogen peroxide can be conventional various concentration, for example, 30% hydrogen peroxide.
In the present invention, the RCA reaction solution can be the reaction solution of commercially available archaeal dna polymerase when institute band, or according to
Reaction solution made of reaction principle configuration (330mM Tris- acetic acid (pH is 7.9 at 37 DEG C), 100mM magnesium acetate, 660mM vinegar
Sour potassium, 1% (v/v) polysorbas20,10mM DTT).
Kit of the invention uses a step competition law, assembles the DNA containing aflatoxin aptamer first
Sensor, the aflatoxin B1 dissociated in sample and aflatoxin aptamer DNA (B) are specifically bound, make its from
It falls off in DNA sensor, to trigger DNA sensor, the amplification of signal is obtained by RCA technology.There are n sections in RCA product chain
It can be aoxidized with catalysis substrate ABTS, from the colourless DNA sequence dna for becoming green.To realize that naked eyes detect.According to competitive principle,
If the free aflatoxin B1 amount in sample is more, the aflatoxin aptamer split away off is more, triggers simultaneously
DNA sensor it is more, and then have more signals.Conversely, then few.The aflatoxin B1 standard items of various concentration gradient are set,
Its signal is detected by the above method, be will test the signal that the aflatoxin B1 of various concentration gradient obtains and is drawn as standard song
Line, hereafter the concentration of aflatoxin B1 is obtained by contrast standard curve in actual sample.
The aflatoxin B1 aptamer, the DNA sensor and the kit of the invention can be applied to
Detect aflatoxin B1.
Specifically, the method for detecting aflatoxin B1 can comprise the following steps that
(1) sample to be tested containing aflatoxin B1 is mixed with aflatoxin B1 extracting solution, is centrifuged and extracts
Clear liquid；
(2) supernatant is mixed and is incubated for DNA sensor solution, archaeal dna polymerase；
(3) hemin solution and HEPES buffer solution is added, be added after incubation at room temperature ABTS reduction-state colorless substrate and
Hydrogen peroxide.
According to the present invention, the weight ratio of the sample to be tested and aflatoxin B1 extracting solution can be 1:1-1000.Step
Suddenly the condition being incubated in (2) can be the incubation conditions of conventional DNA polymerase reaction, for example, 30 DEG C, be incubated for 60min, 90 DEG C
It heats 5min and terminates reaction.The archaeal dna polymerase can be the various archaeal dna polymerases of this field routine, preferably Phi29DNA
Polymerase.The operation instruction when dosage of the archaeal dna polymerase and dNTP can be according to the commercially available enzymes determines.
Principle according to the present invention, those skilled in the art can determine the dosage of each component in step (3), for example, institute
Final concentration of 0.1-1 μM for stating hemin；The final concentration of 1-5mM of the ABTS reduction-state colorless substrate；The end of the hydrogen peroxide
Concentration is 0.05-0.2mM；The HEPES buffer solution is 2 × HEPES buffer solution.On the basis of meeting above-mentioned final concentration, ability
Field technique personnel can according to need the suitable mother liquid concentration of selection.
According to the present invention, measurement aflatoxin B1 is not necessarily to large-scale instrument and equipment, at low cost.Isothermal reaction can be realized
The qualitative detection of naked eyes also can be realized quantitative detection, also, be not necessarily to fluorescent dye, and cost greatly reduces.Use nucleic acid
Aptamers have the characteristics that highly selective as signal receiver.The same of signal is carried out using RCA technology and DNA catalytic sequence
Detection limit (2ppb) greatly reduces in Shi Fang great.The kit is easy to carry, easy to operate.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Specific embodiment
It is further illustrated by the examples that follow the present invention.Wherein, hemin, ABTS reduction-state colorless substrate, Phi29 are poly-
Synthase is commercially available.RCA reaction solution is Phi29 polymeric enzyme reaction buffer.
Preparation example 1: preparation DNA sensor solution.
DNA (A), DNA (B), DNA (C), dNTP and RCA reaction solution are mixed.Wherein, DNA (A), DNA (B), DNA (C),
The concentration of dNTP is respectively 40nM, 60nM, 40nM and 0.5nM.
Wherein, the sequence of DNA (A) are as follows:
5’-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3’。
The sequence of DNA (B) are as follows:
5 '-GTTCCCGTGCTGTTGTCTCTCTGTGTCTGCACGGGTTCGCTAGGCCCACA-3 ' (SEQ ID NO:
7)。
The sequence of DNA (C) are as follows:
5’-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3’。
Embodiment 1
(1) with 70% methanol configure 50 μ l various concentration gradients (concentration be respectively 500 μ g/L, 50 μ g/L, 5 μ g/L,
500ng/L, 50ng/L) aflatoxin B1 standard items；
(2) it is separately added into 50 μ l DNA sensor solution, 10U Phi29 polymerase thereto, mixes, 30 DEG C of incubations
60min, 90 DEG C of heating 5min terminate reaction；
Hemin solution and 2 × HEPES buffer solution is added, be incubated at room temperature after 30min be added ABTS reduction-state colorless substrate and
30% hydrogen peroxide, final concentration of 0.5 μM of the hemin；The final concentration of 2mM of the ABTS reduction-state colorless substrate；It is described
The final concentration of 0.1mM of hydrogen peroxide；Color signal is read after 5min.
Make standard curve.
Embodiment 2
It takes 10g to be detected sample (content of known wherein aflatoxin B1 is 1 μ g/L), 500 μ l aspergillus flavus poison is added
Plain extracting solution (70% methanol) mixes well with it, and centrifugation extracts 50 μ l supernatants；
50 μ l DNA sensor solution, 10U Phi29 polymerase are added into supernatant, mix, 30 DEG C of incubation 60min,
90 DEG C of heating 5min terminate reaction；
Hemin solution and HEPES buffer solution is added, be incubated at room temperature after 30min be added ABTS reduction-state colorless substrate and
30% hydrogen peroxide；Final concentration of 0.5 μM of the hemin；The final concentration of 2mM of the ABTS reduction-state colorless substrate；It is described
The final concentration of 0.1mM of hydrogen peroxide.It visually can be seen that color change after 5min.Color signal is read, is measured according to embodiment 1
Standard curve determine that the content of the aflatoxin B1 of tested sample is 1 μ g/L.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Claims (5)
1. a kind of DNA sensor, which is characterized in that the DNA sensor is made of following three parts:
(1) DNA (A): the circular DNA template being formed by connecting by following DNA sequence dna 5 ' end and 3 ' ends: 5 '-ACTGATATTAAC
CCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3'；
(2) DNA (B): aflatoxin B1 aptamer；The aptamer has following nucleotide sequence: 5 '-GTTCC
CGTGCTGTTGTCTCTCTGTGTCTGCACGGGTTCGCTAGGCCCACA-3'；
(3) DNA (C): RCA primer, the DNA (C) have one of following three kinds of nucleotide sequences:
5'-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3'；
5'-TAATATCAGTCACGGTATTCTTTTCACAACAGCACGGGAAC-3'；
5’-TAATATCAGTCACGGTATTCTTTTCAGAGACAACAGCACGGGAAC-3’。
2. a kind of kit, which is characterized in that the kit includes following components:
(1) aflatoxin B1 extracting solution；
(2) DNA sensor solution；
(3) Phi29 polymerase；
(4) ABTS reduction-state colorless substrate；
(5) hemin solution；
(6) hydrogen peroxide；
(7) HEPES buffer solution；
Wherein, the DNA sensor solution includes following components:
DNA sensor, RCA reaction solution and dNTP as described in claim 1.
3. kit according to claim 2, wherein DNA (A), DNA (B) and DNA (C) in the DNA sensor solution
Concentration be 10-1000nM.
4. kit according to claim 2 or 3, wherein the aflatoxin B1 extracting solution is methanol solution.
5. kit described in any one of DNA sensor described in claim 1 or claim 2-4 is in detection aspergillus flavus
Application in toxin B1.
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| CN107164384B (en) * | 2016-05-26 | 2019-01-04 | 中国农业科学院北京畜牧兽医研究所 | Detect aflatoxin M1Aptamer, sensor, kit and application |
| CN107991293A (en) * | 2017-11-27 | 2018-05-04 | 中山市食品药品检验所 | One kind is used for aflatoxin B1Visible detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517289A (en) * | 2011-11-25 | 2012-06-27 | 国家纳米技术与工程研究院 | Nucleic acid aptamer of aflatoxin B1 and application thereof |
| CN102952802A (en) * | 2012-09-29 | 2013-03-06 | 江南大学 | Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1 |
| CN103725685A (en) * | 2014-01-14 | 2014-04-16 | 厦门大学 | Aptamer AFB1-14 of aflatoxins B1 and application thereof |
| CN104964969A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517289A (en) * | 2011-11-25 | 2012-06-27 | 国家纳米技术与工程研究院 | Nucleic acid aptamer of aflatoxin B1 and application thereof |
| CN102952802A (en) * | 2012-09-29 | 2013-03-06 | 江南大学 | Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1 |
| CN103725685A (en) * | 2014-01-14 | 2014-04-16 | 厦门大学 | Aptamer AFB1-14 of aflatoxins B1 and application thereof |
| CN104964969A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1 |
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