CN109833648B - Vomitoxin and derivative aptamer affinity column thereof, and preparation method and application thereof - Google Patents
Vomitoxin and derivative aptamer affinity column thereof, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an aptamer affinity column for vomitoxin and derivatives thereof, and a preparation method and application thereof. The affinity column takes agarose modified by N-hydroxysuccinimide as a carrier, then the aptamer capable of identifying deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl deoxynivalenol and 15-acetyl deoxynivalenol in a sample with high affinity and high specificity is subjected to covalent coupling with the carrier, and the coupled aptamer composite carrier is filled in the affinity column. The affinity column is mainly used for purifying and purifying vomitoxin and derivatives thereof in other various samples such as food, feed, milk, grain crops, traditional Chinese medicines and the like, so as to be beneficial to high performance liquid chromatography and rapid detection of the toxin in the samples in the later period. 4 toxins can be purified and enriched by one-time column passing, the repeated use times can reach 15 times, the recovery rate is more than 80%, the cost is reduced, and the pretreatment efficiency of the sample is improved.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to an adaptive affinity column for vomitoxin and derivatives thereof, and a preparation method and application thereof.
Background
DON and its derivatives belong to a kind of mycotoxins, also known as vomitoxin, and areTrichothecene toxins are produced mainly by some fusarium species. This class of compounds was isolated in 1973 by Vesonder et al from corn and feed which caused refusal and vomiting in american sows and was named vomitoxin. The trichothecene toxoid has a double bond between C-9 and C-10 in the chemical structure, an oxygen ring at C-12 and C-13, and hydroxyl and acetyl groups at other positions. DON is composed of a 12, 13-epoxy group, an alpha, beta-unsaturated ketone group and 3-OH functional groups, the chemical name of the DON is 3, 7, l5-trihydroxy-12, l 3-epoxy trichothecene-9-ene-8-one (3, 7, l5-trihydroxy-12, 13-epoxytritichotec-9-en-8-one), and the molecular formula of the DON is C5H20O6The relative molecular mass is 296.3, the pure product is colorless needle-shaped crystal, the melting point is (151-153) DEG C, the crystal can be dissolved in water and polar solvents, such as hydrous methanol, hydrous ethanol or ethyl acetate, and the crystal can be stored in the ethyl acetate for a long time. DON has strong heat resistance and acid resistance, is stable at 120 ℃, is stable when the temperature is increased to 180 ℃, and can be damaged when the temperature is increased to 210 ℃ and is continuously heated for 30-40 min. Thus, the DON in the food is not easily destroyed by the food processing during the general cooking process. DON also has strong hiding power, and researches show that the toxicity of diseased wheat is not changed when the diseased wheat is stored in a laboratory for 4 years.
4 toxins of vomitoxin (deoxynivalenol) and deoxynivalenol-3-glucoside (DON-3G), 3-acetyl deoxynivalenol (3-AcDON) and 15-acetyl deoxynivalenol (15-AcDON) which are derivatives of the deoxynivalenol are detected in most samples at the same time, and have high detection rate, and generate combined pollution to crops, and can mutually influence in vivo after eating the crops by mistake. Because the vomitoxin and the derivative thereof have larger harm, a simple, quick, accurate, economic and specific pretreatment method is found, the matrix interference is eliminated, and the method has important significance for researching the pollution condition of the vomitoxin and the derivative thereof.
At present, methods for detecting vomitoxin and derivatives thereof in food, feed and grain samples comprise thin-layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, capillary electrophoresis, liquid chromatography-mass spectrometry and the like. Among them, thin layer chromatography is the first and most widely used method for detecting vomitoxin and its derivatives, and has advantages of being suitable for personnel without special training, low cost, and no need of expensive instrument. However, the sample processing of the thin layer chromatography is complicated, the experimental process is complicated, the required detection period is long, and the interference of impurities is easy to occur. The detection is semi-quantitative by visual inspection, has the defects of large subjective influence, low sensitivity and the like, and can not meet the requirements of modern detection.
The enzyme-linked immunosorbent assay has the advantages of good detection specificity, high sensitivity and lower detection cost, is suitable for screening and general investigation of a large number of samples in basic institutions, and can greatly save time and cost. The major problem of ELISA is that false positives are easily generated. Therefore, the kit is mainly used for screening detection of the basic level.
The instrument analysis methods such as the high performance liquid chromatography, the capillary electrophoresis method, the liquid chromatography-mass spectrometry and the like have the advantages of high accuracy, strong sensitivity, capability of micro-measurement and the like, and are the methods for detecting the toxin in the food which are commonly used at present. However, the requirement for the purity of the sample is high, and some pretreatment processes are required, so that the detection cost is high, the period is long, and the requirement for rapidly screening a large amount of samples cannot be met. The traditional pretreatment technologies include immunoaffinity columns, multifunctional purification columns and the like, and the purification columns are expensive and mostly disposable. Therefore, establishing a high-selection, rapid and effective sample pretreatment technology has become an important problem to be solved urgently in the detection and analysis of vomitoxin and derivatives thereof.
An aptamer is essentially a stretch of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence (10-100 bases) with a specific complex three-dimensional structure and capable of specifically binding to a target. Single-stranded nucleic acid sequences can form secondary structures that provide stringent recognition of bindable ligands and high affinity. By constructing a single-stranded random oligonucleotide library, and carrying out enrichment and screening for multiple times by using a Systematic evolution of ligands by exponential enrichment technology (SELEX), the aptamer with high affinity for a target and specificity is preferably selected in vitro, so that the difficulty caused by in vivo immune reaction is avoided. The aptamer as a novel molecule artificially synthesized in vitro and similar to the antibody function is still in the initial stage of research compared with the mainstream antibody technology, but has shown some advantages different from the antibody, such as consistent batch stability, easy modification, no immunogenicity and the like.
An affinity column based on an aptamer is a novel high-efficiency sample pretreatment technology. The principle is that the target molecule in a complex sample is extracted and purified by utilizing the selective adsorption of an aptamer to the target molecule, and the adsorption is reversible. Affinity column binding of aptamers to conventional instrumentation has become an important development in mycotoxin analysis.
At present, aptamer affinity columns reported at home and abroad only can be used for one toxin, and cannot meet the requirement that 4 toxins including vomitoxin and derivatives thereof are purified and enriched in one step when the vomitoxin and the derivatives thereof exist in a sample in practical application.
Disclosure of Invention
The invention aims to provide an aptamer affinity column for vomitoxin and derivatives thereof, and a preparation method and application thereof.
The invention has the following conception: aiming at the common characteristics of 4 toxins in a practical sample, the invention takes a group common in chemical mechanisms of the four toxins including deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl deoxynivalenol and 15-acetyl deoxynivalenol as a target, and takes an aptamer aiming at the target as an affinity element to prepare a multi-toxin aptamer affinity column suitable for purifying the four toxins including deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl deoxynivalenol and 15-acetyl deoxynivalenol.
In order to achieve the object, the invention provides a DNA aptamer specific to vomitoxin and derivatives thereof, and the nucleic acid sequence of the DNA aptamer is shown as SEQ ID NO. 1.
In the present invention, derivatives of emetic toxins include, but are not limited to, deoxynivalenol-3-glucoside, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol.
In a second aspect, the invention provides a detection reagent, a detection test strip, a detection kit or an affinity column containing the aptamer.
In a third aspect, the invention provides any one of the following uses of the aptamer:
(1) the application in preparing an aptamer affinity column of vomitoxin and derivatives thereof;
(2) the application in preparing detection reagents, detection test strips or detection kits for vomitoxin and derivatives thereof;
(3) the application in the detection of vomitoxin and derivatives thereof.
In a fourth aspect, the invention provides an aptamer affinity column for vomitoxin and derivatives thereof, wherein the filler of the affinity column is obtained by taking agarose modified by N-hydroxysuccinimide as a carrier and then covalently coupling the aptamer of SEQ ID NO. 1 with the carrier.
Preferably, the aptamer is a chemically modified aptamer sequence, the modification including but not limited to amino modification, carboxyl modification, sulfhydryl modification or biotin modification.
More preferably, the aptamer is an amino-modified aptamer sequence, and the modification method is as follows: the C7 indirect arm (- (CH) is covalently linked at the 3 'or 5' end of the aptamer2)7-) or C6 indirect arm (- (CH)2)6-) and then modifying the amino group at the end of the C7 indirect arm or the C6 indirect arm by a covalent bond to give an amino-modified aptamer.
In a fifth aspect, the present invention provides a method for preparing the affinity column, comprising the steps of:
1) preparation of N-hydroxysuccinimide modified agarose: mixing agarose and equal volume of water, adding the mixture into a reactor provided with a pH electrode and a magnetic stirrer, adding N-hydroxysuccinimide into the agarose solution according to the amount of 50mg of N-hydroxysuccinimide per ml of the agarose solution, controlling the pH value of a reaction system to be 9 +/-0.1 (regulating the pH value by NaOH), and reacting for 3-l 2min at the temperature of 20 +/-5 ℃; after the reaction is finished, quickly adding the ice scraps with the same volume into the reaction solution, quickly pouring the mixture into a Buchner funnel, performing suction filtration and washing by using a cold buffer solution with the volume being 10-15 times that of the agarose solution, reacting hydroxyl on the surface of the agarose solution with N-hydroxysuccinimide, and drying to obtain granular N-hydroxysuccinimide modified agarose;
wherein the buffer solution is: 200mM Na2HPO4，5mM MgCl2，pH 8.0；
2) Swelling and washing of the support: taking agarose modified by N-hydroxysuccinimide as a carrier, and soaking 50mg of carrier particles in 1-5mL of 1mM hydrochloric acid for 0.5-1h for swelling; washing the swelled carrier with 1mM hydrochloric acid 1-5mL for 3-6 times, then with distilled water 1-5mL for 2-5 times, and finally with Na2HPO4Washing with buffer solution for 2-5 times, wherein the amount of the buffer solution is 1-5mL each time;
3) aptamer renaturation: dissolving 1-5OD 3 'or 5' end amino modified aptamer in Na2HPO4Renaturation is carried out for 3-5min at the temperature of 75-95 ℃ in 1000 mu L of buffer solution 200-;
4) coupling: adding 1000 mu L of the aptamer solution 200-;
5) and (3) sealing: the coupling product obtained in step 4) was in turn treated with 200mM Na, pH8.02HPO4Washing with aqueous solution for 2-5 times, each time with 1-5mL, then washing with 0.1M Tris-HCl buffer solution with pH8.0 for 2-5 times, each time with 1-5mL, then adding 0.1M Tris-HCl solution with pH8.0 for 2-5mL, shaking at 30 ℃ for reaction for 1-6h, and blocking the remaining active sites to obtain the carrier-aptamer coupling gel;
6) washing: sequentially and alternately washing the carrier-aptamer coupling gel for 3-5 times by using an acetate buffer solution and a Tris-HCl buffer solution, wherein the dosage of the acetate buffer solution or the Tris-HCl buffer solution for each washing is 1-5mL, and removing unconjugated aptamers; resuspending the washed coupling gel with 1-5mL of binding buffer solution to obtain coupling gel suspension ready for column packing;
wherein the acetic acid buffer solution is0.1M acetic acid-sodium acetate aqueous solution containing 0.5M NaCl, pH 4.0; the concentration of the Tris-HCl buffer solution is 0.1M, wherein the Tris-HCl buffer solution contains 0.5M NaCl and has the pH value of 8.0; the binding buffer contained 10mM Tris, 120mM NaCl, 5mM KCl and 5mM MgCl2，pH 7.5；
7) Column assembling: taking a solid phase extraction column with volume of 1-5mL, filling a lower sieve plate, packing the column with the coupling gel suspension until the gel height is 1-2cm, and adding 0.05% w/v NaN30.5-3mL of solution, and storing at 4 ℃;
wherein, the Na is obtained in the steps 2) to 3)2HPO4The buffer solution is as follows: 200mM Na2HPO4，5mM MgCl2，pH 8.0。
Preferably, steps 2) to 7) of the aforementioned method are as follows:
2) ' swelling and washing of the support: taking agarose modified by N-hydroxysuccinimide as a carrier, and soaking 60mg of carrier particles in 2mL of 1mM hydrochloric acid for 1h for swelling; the swollen support was washed 6 times with 1mM HCl 1mL each time, then 2 times with distilled water 1mL each time, and finally with Na2HPO4Washing with 1mL of buffer solution for 2 times;
3) ' aptamer renaturation: dissolving 1OD 3 'or 5' end amino modified vomitoxin and derivative aptamer specific DNA thereof in Na2HPO4Renaturation at 75 deg.C for 5min in 200 μ L buffer solution, and standing at room temperature for 30 min;
4) ' coupling: adding 200 mu L of aptamer solution with good renaturation in the step 3) into the washed carrier in the step 2) and shaking the mixture in a shaking table at 30 ℃ for overnight;
5) ' sealing: step 4)' the resulting coupled product was successively treated with 200mM Na, pH8.02HPO4Washing with aqueous solution for 3 times, 1mL each time, then washing with 0.1M Tris-HCl buffer solution with pH8.0 for 2 times, 1mL each time, then adding 2mL Tris-HCl solution with pH8.0 of 0.1M, shaking in a shaker at 30 ℃ for reaction for 2h, and sealing the residual active sites to obtain the carrier-aptamer coupling gel;
6) ' washing: sequentially and alternately washing the carrier-aptamer coupling gel with acetic acid buffer solution and Tris-HCl buffer solution for 5 times, wherein each timeThe dosage of the secondary washing acetic buffer solution or the Tris-HCl buffer solution is 2mL, and the unconjugated aptamer is removed; resuspending the washed coupling gel with 5mL of binding buffer solution, and preparing the obtained coupling gel suspension for column packing; wherein the acetic acid buffer solution is 0.1M acetic acid-sodium acetate aqueous solution, which contains 0.5M NaCl and has pH of 4.0; the concentration of the Tris-HCl buffer solution is 0.1M, wherein the Tris-HCl buffer solution contains 0.5M NaCl and has the pH value of 8.0; the binding buffer contained 10mM Tris, 120mM NaCl, 5mM KCl and 5mM MgCl2，pH 7.5；
7) ' column installation: taking a solid phase extraction column with volume of 5mL, filling a lower sieve plate, filling the column with the coupling gel suspension until the gel height is 3cm, and adding 0.05% w/v NaN30.5mL of the solution was stored at 4 ℃.
In the invention, the solid phase extraction column and the lower sieve plate are made of materials selected from polypropylene, polystyrene, porous polystyrene or cross-linked porous polystyrene.
Preferably, the aperture of the lower sieve plate is about 10 μm.
In a sixth aspect, the invention provides any one of the following applications of the vomitoxin and derivative aptamer affinity column:
the application of vomitoxin and derivatives thereof in enriching and purifying samples;
② the application in purifying vomitoxin and derivatives thereof in samples.
The sample includes but is not limited to food, feed, milk, food crops and traditional Chinese medicine.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the affinity column has the advantages of simple operation and low price, can simultaneously purify and enrich four vomitoxin and derivatives thereof, and changes the complicated operation that the four toxins need to pass through the four affinity columns respectively in the past. Has the advantages of cost saving, sample saving, high purification efficiency and repeated use. After simple extraction, the sample can be purified on the column, and most of interferents can be removed by one-time purification. The purified extract can be analyzed and detected by instruments such as high performance liquid chromatography. When the actual sample is detected, a plurality of columns are not needed, the pretreatment cost and time are reduced, the pretreatment efficiency is improved, and the method has wide application prospect in basic level and laboratory detection.
The invention fully utilizes the advantages of high specificity and high affinity of the aptamer, and utilizes the specific combination of the vomitoxin and the derivative thereof in the aptamer with the vomitoxin and the derivative thereof in the sample, thereby reducing the cross reaction frequently encountered by the antibody-based immunoaffinity column and greatly improving the purification efficiency of the affinity column. The aptamer is less influenced by the operating environment and organic solvents, and is particularly suitable for purifying lipoid soluble substances of mycotoxin. In contrast, since antibodies in immunoaffinity columns are not resistant to organic solvents, the presence of organic solvents often results in inactivation of the antibodies and a decrease in the efficiency of the column. In addition, immunoaffinity columns are typically disposable because organic solvents may cause inactivation of the antibody. The nucleic acid aptamer affinity column can tolerate organic solvents and can be repeatedly used, and the use cost is greatly reduced.
And thirdly, the aptamer used by the invention is obtained by an in-vitro chemical synthesis method, so that the correctness of the sequence and the consistency among batches can be ensured, and the difference among different batches is greatly reduced. In contrast, different batches of antibodies from different mice or rabbits resulted in large differences in the mass of the antibodies, thereby causing batch-to-batch differences in the mass of the immunoaffinity column.
The (tetra) N-hydroxysuccinimide modified agarose is subjected to covalent coupling, the coupling product is stable, and the coupling rate is high.
And (V) the aptamer affinity column prepared by the invention uses the aptamer to replace an antibody as a recognition element, and has the advantages of low cost, easy storage, stable property and small batch difference compared with the traditional immunoaffinity column. After the sample extracting solution is purified by the aptamer affinity column, the obtained vomitoxin and derivatives thereof have high purity, subsequent purification treatment is not needed, and the vomitoxin and derivatives thereof can be directly used for detection of instruments such as a high performance liquid chromatography and the like, so that the time and the cost of operators are saved.
And sixthly, the aptamer affinity column prepared by the method is mainly used for purifying and purifying vomitoxin and derivatives thereof in other various samples such as food, feed, milk, food crops, traditional Chinese medicines and the like, so that the high performance liquid chromatography and the rapid detection of the vomitoxin in the samples in the later period are facilitated. 4 toxins can be purified and enriched by one-time column passing, the repeated use times can reach 15 times, the recovery rate is more than 80%, the cost is reduced, and the pretreatment efficiency of the sample is improved.
Drawings
FIG. 1 is a schematic structural diagram of an aptamer affinity column for vomitoxin and derivatives thereof prepared in example 1 of the present invention; wherein, 1-an injection port plug; 2-a column; 3-upper screen plate (upper screen plate); 4-a carrier filler; 5-lower sieve plate (lower sieve plate); 6-sample outlet and plugging.
FIG. 2 shows the purification principle of the aptamer affinity column of vomitoxin and its derivatives of the present invention.
FIG. 3 shows the results of the test of the number of times of repeated use and recovery of the aptamer affinity column for vomitoxin and derivatives thereof in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 preparation of aptamer affinity column Using amino-modified vomitoxin and its derivative aptamer
1. Preparation of N-hydroxysuccinimide modified agarose. Agarose Sepharose 4B was used as a carrier, and N-hydroxysuccinimide was used for activation.
Mixing agarose and equal volume of water in a fume hood, adding the mixture into a reactor provided with a pH electrode and a magnetic stirrer, adding N-hydroxysuccinimide into the agarose solution according to the amount of 100 mgN-hydroxysuccinimide per mL of the agarose solution, adjusting the pH value to 11 by using NaOH, controlling the pH value of the whole reaction to be 11, controlling the temperature to be about 20 ℃, and finishing the reaction within 10 min; after completion of the reaction, an equal volume of ice debris was quickly added to the above reaction solution, and quickly poured into a Buchner funnel, and a cold buffer (200mM Na) 10 times the volume of the agarose solution was added2HPO4，5mM MgCl2pH8.0) is filtered and washed, and hydroxyl on the surface of the agarose reacts with N-hydroxysuccinimide to obtain the agarose modified by the N-hydroxysuccinimide.
2. Preparation of amino-modified emetic toxin and its derivative aptamer (SEQ ID NO: 1).
The C7 indirect arm (- (CH) is covalently linked at the 3' end of the aptamer2)7-) and then modifying the amino group at the end of the C7 indirect arm by a covalent bond, thereby obtaining an amino-modified aptamer.
3. Swelling and washing of the support: adding 2mL of 1mM hydrochloric acid into 60mg of N-hydroxysuccinimide modified agarose dry powder, and swelling for 1 h; the swollen support was washed rapidly and repeatedly 6 times with 1mL of HCl (1mM, pH 3.0), 2 times with 1mL of distilled water, and 1mL of Na2HPO4Buffer (200mM Na)2HPO4 5mM MgCl2pH8.0) 2 washes.
4. Aptamer renaturation: 1OD 3' amino group-modified aptamer was dissolved in 200. mu.L of Na2HPO4Buffer (200mM Na)2HPO4 5mM MgCl2pH8.0), renaturation at 75 deg.C for 5min, and standing at room temperature for 30 min.
5. Coupling: 200 mu L of the renatured aptamer solution is added into the washed carrier, and the carrier is shaken on a shaker at 30 ℃ overnight.
6. And (3) sealing: the coupling product was successively treated with 1mL of Na2HPO4(200mM, pH8.0) for 3 times, washing with 1mL of Tris-HCl buffer (0.1M, pH8.0) for 2 times, adding 1mL of Tris-HCl buffer (0.1M, pH8.0), and performing shaking reaction at 30 ℃ for 2h to block the remaining active sites to obtain the carrier-aptamer coupling gel.
7. Washing: the blocked aptamer-carrier conjugate gel was washed with 2mL of acetate buffer (0.1M acetate-sodium acetate, pH4.0, containing 0.5M NaCl) and 2mL of Tris-HCl buffer (0.1M, pH8.0, containing 0.5M NaCl) alternately for 5 times to remove the unconjugated aptamer.
8. Column assembling: the coupling product was treated with 5mL binding buffer (10mM Tris, 120mM NaCl, 5mM KCl, 5mM MgCl)2Ph7.5) and then loaded into an empty SPE cartridge.
1) Empty 1mL SPE cartridges were removed, fitted with a lower sieve plate (10 μm pore size), and then 1mL binding buffer was added to allow to drain naturally.
2) Adding a plug of a lower sample outlet, adding the treated coupling product into an SPE column tube, and standing for 5min to naturally settle the carrier.
3) Add the upper sieve plate (10 μm pore size) and press the sieve plate to bring it above the support.
4) And pulling out the plug of the sample outlet, taking 5mL of binding buffer solution by using an injector, connecting the injector to the sample inlet, and slowly injecting the binding buffer solution into the affinity column until all the liquid is injected into the affinity column, wherein the liquid outlet speed is kept at 1-2 drops/second.
5) Adding 0.05% NaN3(w/v) 0.5mL of buffer solution, and a square injection port plug were added, followed by storage at 4 ℃.
A schematic of the structure of the affinity column prepared in example 1 is shown in FIG. 1. Wherein, 1-an injection port plug; 2-a column; 3-upper screen plate (upper screen plate); 4-a carrier filler; 5-lower sieve plate (lower sieve plate); 6-sample outlet and plugging.
Example 2 purification of emetic toxin and its derivatives and detection in wheat samples Using an aptamer affinity column for emetic toxin and its derivatives
This example quantitatively adds vomitoxin and its derivative standard substance to normal wheat sample, then purifies by using vomitoxin and its derivative aptamer affinity column prepared in example 1, detects by using high performance liquid chromatography-fluorescence detector after purification, and determines recovery rate. The method comprises the following specific steps:
1. wheat sample treatment
1) The wheat samples were crushed.
2) Adding vomitoxin and derivative standard substances thereof into the crushed wheat samples according to the standard of 0.5, 5 and 50 mu g per gram of the sample respectively.
3) 5g of sample was weighed, added with 25mL of methanol-water (7:3, v/v), and placed on a homogenizer for high-speed homogenization at 11000rpm for 3 min.
4) The mixture was filtered through a 0.45 μm syringe filter.
5) 5mL of the filtrate was taken, nitrogen was blown to near dryness at 40 ℃, 0.5mL of methanol-water (7:3, v/v) was added for redissolution, and the volume was made up to 5mL with binding buffer.
6) And (4) taking the complex solution for sample loading purification and detection.
2. And taking out the vomitoxin and the aptamer affinity column of the derivative thereof, opening a sample injection port plug, connecting a sample injection port with a syringe cylinder of the injector, and connecting the injector to the pneumatic control operation frame.
3. The outlet plug was opened, the affinity column was equilibrated with 5mL of binding buffer, and the pressure of the air pump of the air vent manipulator was adjusted to allow the liquid to flow out at a flow rate of 3 drops/sec.
4. Adding the re-solution into the affinity column, and regulating the flow rate to 1-2 drops/second until all the sample flows out of the affinity column.
5. The affinity column was washed with 1mL binding buffer.
6. 1mL of methanol was added and the eluted product was collected.
7. The eluted product is detected by a high performance liquid chromatography-fluorescence detector.
8. The detection result of the high performance liquid chromatography is shown in table 1, and the chromatogram and the detection result of the UPLC-MS/MS are shown in fig. 3.
The purification principle of the vomitoxin and the derivative aptamer affinity column of the invention is shown in figure 2.
TABLE 1 detection results of content HPLC-FLD of vomitoxin and its derivatives in wheat
Therefore, the wheat sample is purified by an aptamer affinity column of the vomitoxin and the derivative thereof, and the vomitoxin can be detected by a high performance liquid chromatography-fluorescence detector (HPLC-FLD). As can be seen from the test results, when 0.5. mu.g, 5. mu.g and 50. mu.g of vomitoxin and derivatives thereof were added to 1g of the pulverized wheat samples, the recovery rate was between 79.23 and 85.78% by using the aptamer affinity column of the present invention.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
Research center of <110> Beijing agriculture quality standard and detection technology
<120> vomitoxin and derivative aptamer affinity column thereof, and preparation method and application thereof
<130> KHP181118791.7
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcccggatcg agcagatatc aagcgcatgg gc 32
Claims (16)
1. The vomitoxin and the derivative specificity DNA aptamer thereof are characterized in that the nucleic acid sequence is shown as SEQ ID NO. 1;
wherein the derivative of vomitoxin comprises deoxynivalenol-3-glucoside, 3-acetyl deoxynivalenol and 15-acetyl deoxynivalenol.
2. A detection reagent comprising the aptamer according to claim 1.
3. A test strip comprising the aptamer of claim 1.
4. A test kit comprising the aptamer according to claim 1.
5. An affinity column comprising the aptamer of claim 1.
6. Use of the aptamer of claim 1 for any of the following:
(1) the application in preparing an aptamer affinity column of vomitoxin and derivatives thereof;
(2) the application in preparing detection reagents, detection test strips or detection kits for vomitoxin and derivatives thereof;
(3) the application in the detection of vomitoxin and derivatives thereof;
wherein the derivative of emetic toxin is as defined in claim 1;
the use is for non-diagnostic and therapeutic purposes.
7. An aptamer affinity column of vomitoxin and derivatives thereof, which is characterized in that the filler of the affinity column is obtained by taking agarose modified by N-hydroxysuccinimide as a carrier and then covalently coupling the aptamer of claim 1 with the carrier;
wherein the derivative of emetic toxin is as defined in claim 1.
8. The affinity column of claim 7, wherein the aptamer is a chemically modified aptamer sequence, and the modification comprises an amino modification, a carboxyl modification, a thiol modification, or a biotin modification.
9. The affinity column of claim 8, wherein the aptamer is an amino-modified aptamer sequence modified by the following method: linking the C7 indirect arm- (CH) at the 3 'or 5' end of the aptamer by a covalent bond2)7Or C6 indirect arm- (CH)2)6Then modifying the amino group by covalent bond at the end of the C7 or C6 indirect arm to give an amino modified aptamer.
10. A method for preparing an affinity column according to claim 9, comprising the steps of:
1) preparation of N-hydroxysuccinimide modified agarose: mixing agarose and equal volume of water, adding the mixture into a reactor provided with a pH electrode and a magnetic stirrer, adding N-hydroxysuccinimide into the agarose solution according to the amount of 50mg of N-hydroxysuccinimide per ml of the agarose solution, controlling the pH value of a reaction system to be 9 +/-0.1, and reacting for 3-l 2min at the temperature of 20 +/-5 ℃; after the reaction is finished, quickly adding the ice scraps with the same volume into the reaction solution, quickly pouring the reaction solution into a Buchner funnel, performing suction filtration and washing by using a cold buffer solution with the volume being 10-15 times that of the agarose solution, and drying to obtain granular N-hydroxysuccinimide modified agarose;
wherein the buffer solution is: 200mM Na2HPO4，5mM MgCl2，pH 8.0；
2) Swelling and washing of the support: taking agarose modified by N-hydroxysuccinimide as a carrier, and soaking 50mg of carrier particles in 1-5mL of 1mM hydrochloric acid for 0.5-1h for swelling; washing the swelled carrier with 1mM hydrochloric acid 1-5mL for 3-6 times, then with distilled water 1-5mL for 2-5 times, and finally with Na2HPO4Washing with buffer solution for 2-5 times, wherein the amount of the buffer solution is 1-5mL each time;
3) aptamer renaturation: dissolving 1-5OD 3 'or 5' end amino modified aptamer in Na2HPO4Renaturation is carried out for 3-5min at the temperature of 75-95 ℃ in 1000 mu L of buffer solution 200-;
4) coupling: adding 1000 mu L of the aptamer solution 200-;
5) and (3) sealing: the coupling product obtained in step 4) was in turn treated with 200mM Na, pH8.02HPO4Washing with aqueous solution for 2-5 times, each time with 1-5mL, then washing with 0.1M Tris-HCl buffer solution with pH8.0 for 2-5 times, each time with 1-5mL, then adding 0.1M Tris-HCl solution with pH8.0 for 2-5mL, shaking at 30 ℃ for reaction for 1-6h, and blocking the remaining active sites to obtain the carrier-aptamer coupling gel;
6) washing: sequentially and alternately washing the carrier-aptamer coupling gel for 3-5 times by using an acetate buffer solution and a Tris-HCl buffer solution, wherein the dosage of the acetate buffer solution or the Tris-HCl buffer solution for each washing is 1-5mL, and removing unconjugated aptamers; resuspending the washed coupling gel with 1-5mL of binding buffer solution to obtain coupling gel suspension ready for column packing;
wherein the acetic acid buffer solution is 0.1M acetic acid-sodium acetate aqueous solution, which contains 0.5M NaCl and has pH of 4.0; the concentration of the Tris-HCl buffer solution is 0.1M, wherein the Tris-HCl buffer solution contains 0.5M NaCl and has the pH value of 8.0; the binding buffer contained 10mM Tris, 120mM NaCl, 5mM KCl and 5mM MgCl2，pH 7.5；
7) Column assembling: taking a solid phase extraction column with volume of 1-5mL, filling a lower sieve plate, packing the column with the coupling gel suspension until the gel height is 1-2cm, and adding 0.05% w/v NaN30.5-3mL of solution, and storing at 4 ℃;
wherein, the Na is obtained in the steps 2) to 3)2HPO4The buffer solution is as follows: 200mM Na2HPO4，5mM MgCl2，pH 8.0。
11. The method as claimed in claim 10, wherein the solid phase extraction column and the lower sieve plate in step 7) are made of polypropylene or polystyrene;
the aperture of the lower sieve plate is 10 mu m.
12. The method as claimed in claim 11, wherein the solid phase extraction column and the lower sieve plate in step 7) are made of porous polystyrene.
13. The method as claimed in claim 11, wherein the solid phase extraction column and the lower sieve plate in step 7) are made of cross-linked porous polystyrene.
14. Use of an affinity column according to any one of claims 7 to 9, wherein:
the application of vomitoxin and derivatives thereof in enriching and purifying samples;
② the application in purifying vomitoxin and derivatives thereof in samples;
wherein the derivative of emetic toxin is as defined in claim 1;
the use is for non-diagnostic and therapeutic purposes.
15. The use of claim 14, wherein the sample comprises food, feed and chinese medicine.
16. Use according to claim 15, wherein the food product comprises milk, food crops.
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