CN102952801A

CN102952801A - Group of oligonucleotide aptamers for specifically identifying aflatoxin B2

Info

Publication number: CN102952801A
Application number: CN2012103701396A
Authority: CN
Inventors: 王周平; 王文凤; 段诺; 吴世嘉; 夏雨; 马小媛
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-09-29
Filing date: 2012-09-29
Publication date: 2013-03-06
Anticipated expiration: 2032-09-29
Also published as: CN102952801B

Abstract

The invention discloses a group of oligonucleotide aptamers for specifically identifying aflatoxin B2. Single-chain DNA (deoxyribonucleic acid) aptamers capable of specifically identifying aflatoxin B2 are obtained through an SELEX (systematic evolution of ligands by exponential enrichment) technology, and can be transferred to report aptamers through a fluorescein label and the like so as to be used for detect aflatoxin B2, and the aptamer sequences have wide application prospect in aspect of accurately and rapidly detecting aflatoxin B2 in food.

Description

One group of specific recognition AFB 2Oligonucleotide aptamer

Technical field

The present invention relates to biological technical field, specially refer to SELEX technology (the Fas lignand system evolution technology of the index concentration) preparation one group and the AFB that utilize in the Protocols in Molecular Biology ₂The nucleic acid aptamer of high specific and high-affinity is for this nucleic acid aptamer is detecting AFB ₂In application scientific basis and theoretical basis are provided.

Background technology

Aflatoxin (aflatoxins, AF) mainly is the secondary metabolic product of Aspergillus flavus and Aspergillus parasiticus bacterium, and its chemical structure is similar, is the derivative of dihydrofuran tonka bean camphor.Determined at present the AFB that has of aflatoxin structure ₁, AFB ₂, AFM ₁Deng 18 kinds, all contain two furan nucleuss and coumarin (having another name called tonka bean camphor) in their basic structure, the former is its toxicity structure, the latter may be carcinogenic relevant with it.Aflatoxin is high temperature resistant, and heat treated is very little to its destruction usually, only just decomposes under melting temperature.Aflatoxin is met alkali and can be decomposed rapidly, but this reaction is reversible, namely restores again under acidic conditions.Allly all may there be aflatoxin in grain and feed in the warm moist climatological region by Aspergillus flavus and Aspergillus parasiticus fungi pollution.Aflatoxin the most easily pollutes peanut, corn, cottonseed, birds, beasts and eggs, meat, Milk and milk products, secondly is wheat, Chinese sorghum and sweet potato, and the aflatoxin-contaminated degree of soybean meal lightly.China's grain and feed are very high by the aflatoxin pollution rate, have brought very large loss for Feed Enterprise and producer.It is 1 class carcinogens that aflatoxin in 1993 delimited by the cancer research mechanism of the World Health Organization (WHO), is the extremely strong highly toxic substance of a kind of toxicity.The hazardness of aflatoxin is that people and animal livers tissue are had destruction, when serious, can cause liver cancer even death.AFB ₂With AFB ₁Structure is extremely similar, only differs from the position of two keys, in view of AFB ₁Strong carinogenicity and toxicity, AFB ₂Also have its character, therefore, how fast, accurately detect AFB ₂Has the important research meaning.

At present, to mycotoxins B ₂Detection method to use maximum be that the antigen-antibody method is immunological method, but this method need to prepare specific antibody, antibody has unstable, the cost of preparation is higher and should not preserve, and is subjected to the restriction of immunogenicity etc.These deficiencies have restricted the application of immunological method in the mycotoxins field.And the detection method detectability that exists at present is generally higher, is not suitable for the mycotoxins poisoning and has the characteristics of disguised and trace.Therefore, set up new for mycotoxins especially AFB ₂For the fast sensitive detection method of Typical Representative particularly necessary.

In the last few years, oligonucleotide aptamer substituted molecule as the prospect of antibody molecule, and its research is comparatively noticeable.Oligonucleotide aptamer is by SELEX process screening and cluster small molecule DNA or RNA fragment target material specific binding.SELEX technology (Systematic Evolution of Ligands by Exponential Enrichment, phyletic evolution index concentration technology) being a kind of new combinatorial chemistry technique of early 1990s development, is a kind of effective ways of studying nucleic acid construct and function.Its ultimate principle is a random single stranded oligonucleotide of external chemosynthesis storehouse, mix with the target material and to hatch, form target material-nucleic acid complexes, the nucleic acid molecule that flush away is not combined with the target material, separate the nucleic acid molecule of being combined with the target material, carry out pcr amplification take this nucleic acid molecule as template, carry out again the screening process of lower whorl.Repeat screening and amplification by the number wheel, obtain at last the oligonucleotide aptamer of high-affinity and high specific, i.e. Aptamer.Utilize pattern and the protein antibodies of the Aptamer identification molecule that the SELEX technology screening obtains similar, but compare with protein antibody, aptamer has more obvious superiority, as not relying on zooblast, not limited by immune condition and immunogenicity, the screening of aptamer is carried out external fully, has time, quality and quantitative selection elasticity, can be when synthetic accurately, fixed point, arbitrarily connect other functional groups and molecule; Aptamer sex change and renaturation are reversible and speed is fast, but Reusability, prolonged preservation and room temperature transportation; Target molecule is wider, outside isolating protein, the Nucleotide macromole, also have small molecules (such as dyestuff, Cocaine, caffeine and theophylline etc.), somatomedin, peptide chain, steroid, carbohydrate, cofactor (such as FMN etc.), even can be used for complete cell, virus, spore etc.; Be combined with target molecule and have stronger specificity and avidity, do not organized or sample in the interference of non-target protein, can be in the situation that the unknown of target character filters out its corresponding aptamer; Aptamer is by occupying target material epi-position, make disease controlled, treatment as clinical medicine, manifested potential application prospect, existing research is arrived the aptamer of respective target material as antagonist by the SELEX technology screening, vascular endothelial growth factor when suppressing tumor growth, thrombus generate the effect of the factor, some toxin proteins etc., to reach therapeutic purpose.Aspect microorganism detection, particularly to some pathogenic bacterias or viral research, although do not know the epi-position of its internal structure, function and these materials, but with it as the target material, screen the aptamer corresponding with it by the SELEX process, detect the target material, become the research and probe focus in this field.

In view of the advantage of SELEX technology with respect to traditional antigen-antibody technology, has higher selectivity, specificity and affinity, and the characteristic of can specification configuration extremely similar material, and the SELEX technology is mainly used in the macromolecular screenings such as whole bacterium and cell at present, in the application in small molecules field especially mycotoxins, seldom, it is reported that the mycotoxins aptamers that filters out only has the aptamer of ochratoxin and fumonisins, if can filter out AFB ₂Aptamer to the detection of mycotoxins important meaning will be arranged, also will promote the SELEX technology in the development in small molecules field simultaneously, also be the perfect of SELEX technology self.

The present invention is with AFB common in food and the grain ₂Be target, utilize the SELEX technology to obtain and AFB ₂The nucleic acid aptamer sequence of specific binding, this sequence can fast, accurately detect AFB ₂, because the stable performance of single strand dna oligonucleotide aptamer, can be directly used in fluorescence or chemoluminescence after synthetic convenient and cheap, modified, chromophoric method detects AFB ₂, therefore simple to operate, direct.This invention can be used widely in fields such as food safety detection, clinical medicine.

Summary of the invention

The object of the invention is to provide a kind of mycotoxins detection method, and particularly a kind of aptamer technology of utilizing fast, accurately detects AFB ₂Method.

The phyletic evolution technology (SELEX technology) of the inventive method utilization index level enrichment part is with AFB ₂Be target, screening obtains the aptamer with target high-affinity specific combination, by Fluoresceincarboxylic acid (FAM) marking method reach fast, AFB in Accurate Diagnosis food or the grain ₂Purpose.

Advantage of the present invention:

(1) compare with the antibody of protein, single stranded oligonucleotide is more stable; Aptamer is external synthetic, mark directly, does not need two of mark to resist, so that operation is more simple, rapid; The synthetic cost of Aptamer is low than the antibody preparation cost, and the cycle is short.

(2) this sequence is from structure significantly, have all the strongest one group of aptamer sequences of the avidity that selects 9 aptamer sequences of different avidity and specificity from target, can the specific recognition AFB ₂

Description of drawings

Fig. 1 is the AFB that the screening of 1-10 wheel obtains ₂-ssDNA denaturing polyacrylamide electrophorogram

Fig. 2 is AFB ₂The saturated binding curve figure of-aptamer

Fig. 3 is AFB ₂-aptamer specificity figure

Table 1 represents aptamer and AFB ₂Dissociation constant

Embodiment:

The below is by SELEX technology screening specific combination part aptamer and AFB ₂Nucleic acid aptamer preparation method and Rapid Detection part aptamer and the AFB of dissociation constant ₂The application of dissociation constant aspect.

1, synthetic random single-stranded DNA banks and primer (IDT company is synthetic)

Random single chain DNA (ssDNA) library: 5 '-AGC AGC ACA GAG GTC AGA TG (N40) CCT ATGCGT GCT ACC GTG AA-3', having made up length is the ssDNA pool of 80nt, two ends are the immobilized primer sequence, the centre is the stochastic sequence of 40 bases, and storage capacity is more than 1014; Primer I: 5 '-AGCAGCACAGAG GTCAGATG-3 '; Primer I I:5 '-PO ₄-TTC ACG GTA GCA CGC ATA GG-3 ' all is mixed with ℃ storage of 100 μ mol/L stock solutions-20 with the TE damping fluid with ssDNA pool and two kinds of primers for subsequent use.

2, the pcr amplification in double-stranded DNA (dsDNA) and single stranded DNA (ssDNA) library

Get 2nmol ssDNA pool (second takes turns the each 200pmol of adding of beginning) and add 500 μ L binding buffer liquid BB (pH7.0:100mmol/L NaCl, 20mmol/L Tris-HCl pH7.6,2mmol/L MgCl ₂, 5mmol/L KCl, 1mmol/L CaCl ₂, 0.02%Tween20), then 95 ℃ of water-bath 5min, ice bath 5min get 200 μ L and are connected with target substance AFB ₂Magnetic bead, in advance with BB flushing, with magnet it is adsorbed on the pipe bottom and abandons supernatant, then the ice bath product is added wherein 37 ℃ of shaking tables hatching 2h.To hatch system and shake 20s with vortex mixer, and add magnet and abandon supernatant, repeatedly with BB flushing 5 times, each 1mL.In magnetic bead, add elution buffer 1 * pcr amplification damping fluid 100 μ L, 95 ℃ of water-bath 5min, ice bath 5min shakes rapidly 20s and adds magnet the supernatant sucking-off is elutriant, adds 100 μ L1 * pcr amplification damping fluid again and repeat wash-out in magnetic bead.With twice elutriant mixing as template PCR application of sample: template 5 μ L, each 1 μ L of upstream and downstream primer, dNTP1 μ L, 10 * pcr amplification damping fluid, 5 μ L, aqua sterilisa 36.5 μ L, Taq enzyme 0.5 μ L, cumulative volume are that 50 μ L are at 95 ℃ of 5min, 95 ℃ of 30s, 60 ℃ of 40s, 72 ℃ of 30s circulate 30 times, 72 ℃ of 7min increase under 12 ℃ of 2min conditions.With amplified production with 6 * dna gel sample-loading buffer (tetrabromophenol sulfonphthalein 0.25%, sucrose 40%, water 59.75%) run denaturing polyacrylamide gel electrophoresis, whether the checking amplification is successful, then under similarity condition, carry out the batch amplification in order to purifying and strand preparation, the strand that enzyme is cut is put in 4 ℃ of environment and is stored for future use, and carries out the screening of next round.

3, screen used AFB ₂Obtain and process

AFB ₂Belong to small molecules, be combined with the SELEX technology and must use certain carrier, at first selected amidized magnetic bead as carrier, then to AFB ₂Carry out activation and made it be with carboxyl, adopted the EDC-NHS method of attachment with AFB ₂Be fixed on the surface of magnetic bead, store for future use in 4 ℃ of environment after the sign activation successful connection.

4, Cloning and sequencing

With the tenth ssDNA library of taking turns enrichment, pcr amplification is double-stranded, serves sea living worker Bioisystech Co., Ltd and carries out determined dna sequence, obtains 30 aptamer sequences.

5, the aptamer sequential structure is analyzed

Adopt DNAMAN software and RNA Structure4.2 software respectively the aptamer sequence to be carried out primary structure and secondary structure analysis, obtain homology information and the secondary structure collection of illustrative plates of 30 sequences.In conjunction with primary structure homology and secondary structure sequence is divided into 8 families, selects 1 Stability Analysis of Structures from each family, the sequence that energy level is lower is that next step evaluation is carried out in representative, totally 8.

6, aptamer and AFB ₂Avidity and specific assay

6.1 aptamer avidity is analyzed

According to AFB ₂The I and II constitutional features of-aptamer, all sequences that order-checking is obtained divides into groups, and picks out energy level lower from every group, and the sequence that structure is more stable is served Hai Shenggong synthetic and at its 5 ' end mark vitamin H.

With 9 synthetic AFB that indicate vitamin H ₂The magnetic bead of-aptamer and avidin is assembled into the aptamers probe, with BB be diluted to respectively 10,20,40,60,80,100,120,150nmol/L, in the solution of above-mentioned different concns aptamer, add AFB again ₂(the AFB in the application ₂With methyl alcohol-BB dissolving), make AFB ₂Final concn be 1000ng/L, 37 ℃, 3000rpm shaking table are hatched 1h, magnetic divides the abandonment supernatant, repeatedly wash 5 times with BB, at excitation wavelength 375nm, emission wavelength 425mm surveys the fluorescence intensity on the magnetic bead under the voltage 800V, parallel three times, each represents the dissociation constant K of sequence to use GraphPad Prism5 computed in software _dValue, and draw variant concentration aptamer to AFB ₂Saturation curve, the result is as shown in Figure 2.

Table 1

6.2 specificity analyses

Analytical results according to 6.1 is picked out and AFB ₂3 aptamers (1,17,25) that avidity is stronger carry out specificity analyses, at first each aptamer are assembled into probe, get this probe 50pmol, respectively with the AFB of isodose ₂, AFB ₁, AFG ₁, AFG ₂Hatch, 37 ℃, 3000rpm shaking table are hatched 1h, add magnetic field and abandon supernatant, repeatedly wash 5 times with BB, survey the fluorescence intensity in the washing lotion, parallel three times.Can be reached a conclusion by Fig. 3: aptamers (1,17,25) and AFB ₂Combination rate all apparently higher than AFB ₁, AFG ₁And AFG ₂, show aptamers (1 (in the sequence table 1), 17 (in the sequence table 2), 25 (in the sequence table 3)) but specific recognition AFB ₂

Sequence table

Claims

1. the oligonucleotide aptamer of one group of specific recognition AFB 2, its oligonucleotide sequence is:

1) sequence shown in the sequence 1,2,3 in the sequence table;

2) sequence with sequence 1 same function in the sequence table that the nucleotide sequence shown in the sequence 1 forms through replacing, lack and/or insert one or several Nucleotide in the sequence table;

3) sequence with sequence 2 same functions in the sequence table that the nucleotide sequence shown in the sequence 2 forms through replacing, lack and/or insert one or several Nucleotide in the sequence table;

4) sequence with sequence 3 same functions in the sequence table that the nucleotide sequence shown in the sequence 3 forms through replacing, lack and/or insert one or several Nucleotide in the sequence table.

2. oligonucleotide aptamer claimed in claim 1 application aspect the AFB 2 in detecting food.

3. the application described in according to claim 2 is characterized in that 5 ' end of described oligonucleotide aptamer or 3 ' end can pass through FITC, or FAM, or vitamin H, or digoxigenin labeled.