AU2019101761A4 - An aptamer affinity column of Alternariol and preparation method and application thereof - Google Patents

Abstract

The invention provides an aptamer affinity column for Alternariol and preparation method and use hereof The affinity column uses NHS-activated sepharose as a carrier, and then a nucleic acid aptamer :apable of high-affinity and high-specific recognition of Alternariol is covalently coupled to the carrier, ind the coupled product is used as a packing to pack the affinity column. The affinity column of the >resent invention can be used repeatedly for at least 20 times, and the sample volume can reach 30 mL. It tas a good purification and enrichment effect on Alternariol, and is mainly used for purification and nrichment of Alternariol toxin in fruits, vegetables, feed, food, food crops and other complex samples , o as to facilitate the later high-performance liquid chromatography and rapid detection of the toxin in the ample, which has broad application prospects.

Description

An aptamer affinity column of Alternariol and preparation method and application thereof

Technical field

The invention belongs to the technical field of food safety detection, and specifically relates to an

iltemator aptamer affinity column of Alternariol and preparation method and application thereof

background art

Alternariol (alternaria toxin, AOH) is a secondary metabolite produced by Alternaria. It is a toxin

ubiquitous in fruits, vegetables and field crops. Alternaria can grow and reproduce in low temperature and

tumid environment. Therefore, the contamination of Alternariol toxin is common in the samples of fruits,

vegetables, grains, etc. that rot during cold storage or long-distance transportation. AOH has chronic or

cute toxicity such as mutagenicity, carcinogenicity and teratogenicity to humans or livestock. AOH has

>otential genotoxicity. When the concentration of AOH is >1 M, it is more damaging to DNA in human

:ells. The mycotoxin properties of AOH can significantly inhibit the activity of topoisomerase IIa and

nake DNA strand breaks. Therefore, the research on Alternaria toxin has attracted the attention of

cientists. The European Union has released scientific opinions on the health risks of Alternaria toxins in

ood and feed to humans and animals. Because the chemical properties of Alternariol are relatively stable,

t is difficult to remove even during high-temperature cooking during processing and storage, making the

brmation of the toxin in daily life unavoidable. Moreover, micropollution or trace pollution can cause

erious toxicity, and therefore there is a need for high sensitivity, accurate and reliable method for

Detection.

The current detection methods of Alternariol in food, feed and grain samples include thin-layer

Thromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, capillary electrophoresis, liquid-mass spectrometry, etc. Enzyme-linked immunosorbent assay has the

advantages of good detection specificity, high sensitivity, and low detection cost. It is suitable for

screening and census of a large number of samples in basic institutions, which can greatly save time and

cost. The main problem of the enzyme-linked immunosorbent assay is that it is easy to cause false

positives, which is mainly related to the low antibody titer and the difficulty in preparing small molecule

toxin antibodies.

Instrumental analysis methods such as high performance liquid chromatography, capillary

electrophoresis and liquid mass spectrometry, which have the advantages of high accuracy, strong

sensitivity, and enablement of micro-determination, are currently commonly used methods for the

detection of toxins in food. However, because of its high requirements for sample purity, some pre-treatment processes are required and result in high detection costs and long cycles, which cannot meet he requirements for rapid screening of large quantities of samples. Traditional pretreatment technologies include immunoaffinity columns, multi-ffunctional purification columns, etc. These purification columns re expensive and mostly disposable. Therefore, the establishment of a highly selective, fast and effective ample pretreatment technology has become an important issue that needs to be resolved in the detection nd analysis of Alternariol.

An aptamer is essentially a fragment of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)

equence (10-100 bases) that has a specific complex three-dimensional structure and can specifically bind

o the target. The single-stranded nucleic acid sequence can form a secondary structure, which has strict

ecognition ability and high affinity for binding ligands. By constructing a library of single-stranded

andom oligonucleotides, Systematic evolution of ligands by exponential enrichment (SELEX) is used for

nultiple enrichment and screening, nucleic acid aptamers with specificity and high affinity for the target

vere screened out in vitro, thus avoiding the difficulties caused by in vivo immune response. Aptamers

ire a new type of molecules that are artificially synthesized in vitro and have similar functions to

antibodies. Compared with mainstream antibody technology, its research is still in the early stage, but it

tas shown some advantages that are different from antibodies, such as consistent stability in batches, ease

o modify, and no immunogenicity, etc.

Affinity column based on aptamer is a new and efficient sample pretreatment technology. Its

principlee is to use the selective adsorption of aptamers to target molecules to achieve the extraction and

urification of target molecules in complex samples, and this adsorption is reversible. The combination of

_ptamer affinity column and conventional instrument analysis has become an important development

direction in mycotoxins analysis.

At present, aptamer affinity columns reported in China and abroad are mainly for Ochratoxin, and it

is difficult to meet the requirements for enrichment/purification of Alternariol in samples in practical

applications. There is no report about the aptamer affinity column of Alternariol in China and abroad.

Summary of the invention

The object of the present invention is to provide an aptamer affinity column of Alternariol and

preparation method thereof

Another object of the present invention is to provide a use of the aptamer affinity column of

Alternariol in enrichment and purification, and purification of Alternariol in samples.

The concept of the present invention is given below: a high-specificity and high-affinity Alternariol aptamer is modified with an amino group through the C7 or C6 indirect arm and then is covalently

:oupled with the N-hydroxysuccinimide modified carrier. After washing and blocking, a specific affinity

:olumn packing of Alternariol is obtained. After column packing, a high-affinity aptamer affinity column

sprepared.

In order to achieve the object of the present invention, in a first aspect, the present invention

>rovides an Alternariol-specific DNA aptamer, with a nucleic acid sequence shown in SEQ ID NO:1.

In a second aspect, the present invention provides a detection reagent, a detection test strip, a

etection kit or an affinity column containing the aptamer.

In a third aspect, the present invention provides any of the following use of the aptamer:

(1) use in the preparation of aptamer affinity column of Alternariol;

(2) use in the preparation of test reagent, test strip or test kit for Alternariol;

(3) use in the detection of Alternariol.

In a fourth aspect, the present invention provides an aptamer affinity column of the Alternariol. The

>acking of the affinity column is obtained by using NHS-activated sepharose as a carrier and covalently

:oupling the carrier with the aptamer shown in SEQ ID NO:1.

Preferably, the aptamer is a chemically modified aptamer sequence, and the modification methods

nclude but are not limited to amino modification, carboxyl modification, sulfhydryl modification or

>iotin modification.

More preferably, the aptamer is an amino-modified aptamer sequence, and the modification method

s given below: the 3' or 5' end of the nucleic acid aptamer is covalently bonded to C7 indirect arm

(CH 2 ) 7 - or C6 indirect arm -(CH 2) 6-, and then the end of C7 indirect arm or C6 indirect arm is modified

with amino group through covalent bond to obtain an amino-modified aptamer.

In a fifth aspect, the present invention provides a method for preparing the affinity column,

including the following steps:

1) Preparation of NHS (N-hydroxysuccinimide)-activated sepharose

4 10-15 mL sepharose gel 4FF is washed with water and vacuum filtered, washed with 30-50% and

-70% acetone (preferably 5-8 times the volume of sepharose gel) in a funnel (preferably a sand core

funnel), and finally washed with dioxane (preferably 5-8 times the volume of sepharose gel, analytically

pure) 5-8 times, vacuum filtered and transferred to a triangular flask (preferably a 100 mL triangular flask

with a stopper), and then 10-15mL dioxane, 100-300pL allyl glycidyl ether and 300-1000 L boron

trifluoride ethyl ether, and shaked at 120-180 rpm and 30-40°C (preferably at 140 rpm and 35°C) for

-60 min in a shaker (preferably in water bath); the resulting matrix after reaction is washed with

-50% and 5 0 -7 0 % acetone (preferably 5-8 times the volume of sepharose gel) in sequence, and finally

vashed with deionized water to obtain an activated matrix;

@carboxylation of thioglycolic acid: 1.20-2mL thioglycolic acid, 10-15mL deionized water and

).25-0.5g ammonium persulfate are added to 10-15mL activated matrix, and reacted at 60-80°C for 8-12

i to obtain a thioglycolic acid carboxylated sepharose gel;

@ coupling of NHS groups: 10-15 mL thioglycolic acid carboxylated sepharose gel is washed with

;0-50%, 70-90% and 100% acetone solution, and then washed with dioxane several times (preferably 5-8

imes) to obtain a dioxane-modified sepharose gel; the dioxane-modified sepharose gel is transferred to a

riangular flask (preferably a 100 mL triangular flask with stopper), and 10-15mL dioxane, 1-5g NHS and

-5g N,N-dicyclohexylcarbimide (DCC) are added, shaked and reacted at 25-30°C for 8-12h; finally, the

natrix is washed with dioxane, methanol, and acetone in sequence to obtain a NHS-activated sepharose

as a carrier) that will be stored in isopropanol away from light.

2) Preparation of amino-modified aptamer of Alternariol

the 3' end of the nucleic acid aptamer is covalently bonded to C7 indirect arm -(CH 2) 7-, and then the

nd of the C7 indirect arm is covalently modified with an amino group to obtain an amino-modified

ptamer;

3) Washing of NHS-activated sepharose: 300-500 pL the NHS-activated sepharose was added in a

entrifuge tube (preferably a centrifuge tube with a volume of 1.5-2 mL) and washed with 1-1.5 mM

preferably 1 mM) hydrochloric acid several times (preferably 2-5 times), 1-2 mL each time to obtain a

washed carrier ;

4) Renaturation of the aptamer: 1-5 OD amino-modified aptamer of Alternariol is dissolved in

200-1000 L Na 2HPO4 buffer, renaturated at 75-95°C for 3-5 min, and then placed at room temperature

for 15-60 min to obtain a renaturated aptamer solution; wherein the Na2HPO 4 buffer is: 200 mM

Na 2HPO4, 5mM MgCl2, pH 8.0;

5) Coupling: 200-1000 L renaturated aptamer solution in step 4) is added to the washed carrier in

step 3), and shaked overnight at 25-35°C (preferably 30°C) in a shaker;

6) Blocking: After centrifugation of the obtained coupling product in Step 5) to remove the

supernatant, 1-5 mL blocking buffer (0.2% BSA, 0.1M MES, 0.15M NaCl, pH6.0) is added, and shaked

at 30-40°C for 2-5 hours in a shaker to block the remaining active sites to obtain a carrier-aptamer

coupling gel;

7) Washing: the above-mentioned carrier-aptamer coupling gel is washed with washing buffer

'50mM Tris-HCl, 0.15M NaCl, pH 7.2) several times (preferably 3-5 times) to remove uncoupled

iptamers; the washed coupling gel is resuspend with 1-5 mL of binding buffer (10 mM Tris HCl, 120 mM

4aCl, 5 mM KCl, 1mIM MgC2), and the resulting coupling gel suspension is ready to be packed into the

:olumn;

8) Packing the column: a solid phase extraction column with a volume of 1-5 mL is padded well with

lower sieve plate, and the column is packed with the above coupling gel suspension until the gel height

s 1-2 cm (preferably about 1 cm), and 0.5-3 mL of 0.02-0.05% w/v (preferably 0.05% w/v) NaN 3

olution is added, and stored at 4-10°C (preferably 4C).

Preferably, steps 1)-8) in the foregoing method are given below:

1) Preparation of NHS (N-hydroxysuccinimide)-activated sepharose

( 10 mL sepharose gel 4FF is washed with water and vacuum filtered, washed with 30% and 70%

cetone (5 times the volume of sepharose gel) in a sand core funnel, andfinally washed with 100%

ioxane (5 times the volume of sepharose gel) 5 times, vacuum filtered and transferred to a 100 mL

riangular flask with a stopper, and then 10 mL dioxane, 100 pL allyl glycidyl ether and 300 L boron

rifluoride ethyl ether are added, and shaked at 140 rpm and 35 °C for 45 min in a shaker in water bath;

he resulting matrix after reaction is washed with 30% and 70% acetone (5 times the volume of sepharose

;el) in sequence, and finally washed with large amount of deionized water to obtain an activated matrix; @carboxylation of thioglycolic acid: 1.20 mL thioglycolic acid, 10 mL deionized water and 0.25 g

ammonium persulfate are added to 10 mL activated matrix, and reacted at 60 °C for 8 h to obtain a

.hioglycolic acid carboxylated sepharose gel;

@ coupling of NHS groups: 10 mL thioglycolic acid carboxylated sepharose gel is washed with

%, 90% and 100% acetone solution, and then washed with 100% dioxane 5 times to obtain a

dioxane-modified sepharose gel; the dioxane-modified sepharose gel is transferred to a 100 mL triangular

flask with stopper, and 10 mL dioxane, 1 g NHS and 1 g DCC are added, shaked and reacted at 25 °C

for 8 h; finally, the matrix is washed with large amount of dioxane, methanol, and acetone in sequence to

obtain a NHS-activated sepharose as a carrier that will be stored in isopropanol away from light.

2) Preparation of amino-modified aptamer of Alternariol

the 3' end of the nucleic acid aptamer (SEQ ID NO:1) is covalently bonded to C7 indirect arm

-(CH 2 ) 7-, and then the end of the C7 indirect arm is covalently modified with an amino group to obtain an

amino-modified aptamer;

3) Washing of NHS-activated sepharose: 300 pL the NHS-activated sepharose is added in a

:entrifuge tube with a volume of 1.5 mL and washed with 1 mM hydrochloric acid 2 times, 1 mL each

ime, to obtain a washed carrier ;

4) Renaturation of the aptamer: 1 OD amino-modified aptamer of Alternariol is dissolved in500 L

4a 2 HPO4 buffer, renaturated at 95°C for 5 min, and then placed at room temperature for 30 min to obtain

- renaturated aptamer solution;

5) Coupling: 500 L renaturated aptamer solution in step 4) is added to the washed carrier in step 3),

ind shaked overnight at 30°C in a shaker;

6) Blocking: After centrifugation of the obtained coupling product in Step 5) to remove the

upernatant, 1 mL blocking buffer is added, and shaked at 30 °C for 2 hours in a shaker to block the

emaining active sites to obtain a carrier-aptamer coupling gel;

7) Washing: the above-mentioned carrier-aptamer coupling gel is washed with washing buffer 5

imes to remove uncoupled aptamers; the washed coupling gel is resuspended with 1 mL of binding buffer,

ind the resulting coupling gel suspension (as column packing) is ready to be packed into the column;

8) Packing the column: the above coupling gel suspension is resuspended with 5 mL binding buffer,

ind then packed into an empty SPE column tube.

(1) an empty 1 mL SPE column tube is padded well with a lower sieve plate (aperture size 10 pm),

ind then 1 mL binding buffer is added to allow it to empty out naturally.

(2) with addition of a plug of the lower sample outlet, the coupling gel suspension is added to the

;PE column tube until the gel height is about 1 cm, and the tube is let stand for 5 min to allow the carrier

to settle naturally.

(3) an upper sieve plate (aperture size 10 m) is added, and pressed to allow it reach the top of the

carrier.

(4) the plug of the sample outlet is pulled out, and a syringe is used to take 5 mL of binding buffer,

and connected to the injection port to slowly inject the binding buffer into the affinity column, and the

output rate is kept at 1-2 drops /sec until all the liquid is injected into the affinity column.

(5) 0.5 mL of 0.05% NaN 3 (w/v) buffer is added, a plug of the upper injection port is added, and

then the column is stored in a refrigerator at 4°C.

In the present invention, the material of the solid phase extraction column and the lower sieve plate

is selected from materials such as polypropylene, polystyrene, porous polystyrene or cross-linked porous

polystyrene.

Preferably, the aperture size of the lower sieve plate is about 10 [m.

In the sixth aspect, the present invention provides any one of the following use of the aptamer

ffinity column of Alternariol:

Use of the affinity column according to any one of claims 4-6:

0 use in the enrichment and purification of Alternariol in samples;

@ use in the purification of Alternariol in samples.

The samples include, but are not limited to, food, feed, grain crops, traditional Chinese medicine,

ind other complex samples.

The schematic diagram of the structure of the aptamer affinity column of Alternariol of the present

invention is shown in FIG. 1, and its working principle is shown in FIG. 2.

With the above technical solutions, the present invention has at least the following advantages and

>eneficial effects:

(1) The aptamer affinity column of Alternariol of the present invention is simple to prepare and low

n price. The sample volume can reach 30 mL. The affinity column can be used repeatedly for at least 20

imes and the recovery rate is over 85%, which reduces the cost and improve the sample pretreatment

ficiency. It has the advantages of saving cost, saving samples, high purification efficiency and

epeatable use. After simple extraction, the sample can be purified on the column. One purification can

emove most of the interfering substances. The purified extract can be analyzed and tested with

instruments such as high performance liquid chromatography.

(2) The present invention makes full use of the advantages of high specificity and high affinity of

aucleic acid aptamers, and utilizes the Alternariol aptamer to specifically bind to the Alternariol in the

samples, which reduces the frequent cross-reactions of antibody-based immunoaffinity columns and

greatly improves the purification efficiency of the affinity column. Nucleic acid aptamers are less affected

by the operating environment and organic solvents, and are especially suitable for the purification of

mycotoxins lipid-soluble substances. In contrast, since the antibodies in the immunoaffinity column are

resistant to organic solvents, the presence of organic solvents often causes the inactivation of the

antibodies and reduces the efficiency of the affinity column. In addition, because organic solvents may

cause antibody inactivation, immunoaffinity columns are usually for single use. The nucleic acid aptamer

affinity column can tolerate organic solvents and can be used repeatedly, which greatly reduces the use

cost.

(3) The nucleic acid aptamer provided by the present invention is obtained by in vitro chemical synthesis. Compared with the traditional immunoaffinity column, the nucleic acid aptamer instead of antibody as the recognition element can ensure the correctness of the sequence and the consistency

>etween batches, greatly reducing the difference between different batches. In contrast, different batches

>f antibodies come from different mice or rabbits, resulting in large differences in the quality of the

antibodies and subsequent a batch-to-batch difference in the quality of the immunoaffinity column.

(4) The N-hydroxysuccinimide activated sepharose is subjected to a covalent coupling, the coupling

>roduct is stable, and the coupling rate is high.

(5) The present invention utilizes the secreened Alternariol specific nucleic acid aptamer as an

ffinity element to prepare an aptamer affinity column for purification and enrichment of Alternariol. The

emoval of impurities in the sample before the actual sample detection reduces the cost and time of the

>retreatment, and improves the efficiency of the pretreatment. It has a wide range of application prospects

n the basic level and laboratory testing.

(6) After the sample extract is purified by the aptamer affinity column of the present invention, the

>btained Alternaria sol is of high purity, and with no other purification treatment, it can be directly used

or detection by high-performance liquid chromatography and other instruments, saving operation time

ind expense.

)escription of the drawings

Figure 1 shows a schematic diagram of the structure of an aptamer affinity column of Alternariol

>repared in Example 1 of the present invention; wherein, 1-injection port plug; 2-column; 3-upper sieve

>late; 4-carrier packing; 5-lower sieve plate; 6-sample outlet plug.

Figure 2 shows the purification principle of the aptamer affinity column of Alternariol of the present

invention.

Figure 3 shows the test results of the number of times of repeated use and the recovery rate of the

aptamer affinity column of Alternariol in Example 2 of the present invention.

Figure 4 shows the secondary structure of the library designed in Example 3 of the present

invention.

Figure 5 shows the results of the quality of the established library detected by flow cytometry in

Example 3 of the present invention.

Figure 6 shows the Q-PCR results of the sample single-stranded amplification in Example 3 of the

present invention.

Figure 7 shows the results of the AOH aptamer affinity determination in Example 3 of the present invention.

Figure 8 shows the results of specificity detection of aptamers in Example 3 of the present

invention.

Figure 9 shows the structural difference between AOH and its structural analogue AME in Example

of the present invention.

Figure 10 shows the AOH specificity detection results with AME as the interference substance in

examplee 3 of the present invention.

Embodiments for carrying out the modes

The following examples are used to illustrate the present invention, but not to limit the scope of the

>resent invention. Unless otherwise specified, the technical means used in the examples are conventional

neans well known to those skilled in the art, and the raw materials used are all commercially available

>roducts.

Example 1 Preparation of aptamer affinity column using amino-modified Alternariol aptamer

1) Preparation of NHS-activated sepharose

( 10 mL sepharose gel 4FF was washed with water and vacuum filtered, washed with 30% and

'0% acetone (5 times the volume of sepharose gel) in a sand core funnel, and finally washed with 100%

ioxane (5 times the volume of sepharose gel) 5 times, vacuum filtered and transferred to a 100 mL

riangular flask with a stopper, and then 10 mL dioxane, 100 pL allyl glycidyl ether and 300 L boron

rifluoride ethyl ether were added, and shaked at 140 rpm and 35 °C for 45 min in a shaker in water bath;

he resulting matrix after reaction was washed with 30% and 70% acetone (5 times the volume of

-epharose gel) in sequence, and finally washed with large amount of deionized water to obtain an

activated matrix;

@carboxylation of thioglycolic acid: 1.20 mL thioglycolic acid, 10 mL deionized water and 0.25 g

ammonium persulfate were added to 10 mL activated matrix, and reacted at 60 °C for 8 h to obtain a

thioglycolic acid carboxylated sepharose gel;

@ coupling of NHS groups: 10 mL thioglycolic acid carboxylated sepharose gel was washed with

%, 90% and 100% acetone solution, and then washed with 100% dioxane 5 times to obtain a

dioxane-modified sepharose gel; the dioxane-modified sepharose gel was transferred to a 100 mL

triangular flask with stopper, and 10 mL dioxane, 1 g NHS and 1 g DCC were added, shaked and

reacted at 25 °C for 8 h; finally, the matrix was washed with large amount of dioxane, methanol, and

acetone in sequence to obtain a NHS-activated sepharose as a carrier that will be stored in isopropanol away from light.

2) Preparation of amino-modified aptamer of Alternariol

the 3' end of the nucleic acid aptamer (SEQ ID NO:1) was covalently bonded to C7 indirect arm

(CH 2 ) 7 -, and then the end of the C7 indirect arm was covalently modified with an amino group to obtain

in amino-modified aptamer;

3) Washing of NHS-activated sepharose: 300 pL the NHS-activated sepharose was added in a

:entrifuge tube with a volume of 1.5 mL and washed with 1 mM hydrochloric acid 2 times, 1 mL each

ime, to obtain a washed carrier ;

4) Renaturation of the aptamer: 1 OD amino-modified aptamer of Alternariol was dissolved in500

tL Na2HPO4 buffer, renaturated at 95°C for 5 min, and then placed at room temperature for 30 min to

>btain a renaturated aptamer solution;

5) Coupling: 500 L renaturated aptamer solution in step 4) was added to the washed carrier in step

),and shaked overnight at 30°C in a shaker;

6) Blocking: After centrifugation of the obtained coupling product in Step 5) to remove the

upernatant, 1 mL blocking buffer was added, and shaked at 30 °C for 2 hours in a shaker to block the

emaining active sites to obtain a carrier-aptamer coupling gel;

7) Washing: the above-mentioned carrier-aptamer coupling gel was washed with washing buffer 5

imes to remove uncoupled aptamers; the washed coupling gel was resuspended with 1 mL of binding

>uffer, and the resulting coupling gel suspension (as column packing) was ready to be packed into the

:olumn;

8) Packing the column: the above coupling gel suspension was resuspended with 5 mL binding

buffer, and then packed into an empty SPE column tube.

(1) an empty 1 mL SPE column tube was padded well with a lower sieve plate (aperture size 10 m),

and then 1 mL binding buffer was added to allow it to empty out naturally.

(2) with addition of a plug of the lower sample outlet, the coupling gel suspension was added to the

SPE column tube until the gel height was about 1 cm, and the tube was let stand for 5 min to allow the

carrier to settle naturally.

(3) an upper sieve plate (aperture size 10 m) was added, and pressed to allow it reach the top of the

carrier.

(4) the plug of the sample outlet was pulled out, and a syringe was used to take 5 mL of binding

buffer, and connected to the injection port to slowly inject the binding buffer into the affinity column, and the output rate was kept at 1-2 drops /sec until all the liquid was injected into the affinity column.

(5) 0.5 mL of 0.05% NaN 3 (w/v) buffer was added, a plug of the upper injection port was added,

nd then the column was stored in a refrigerator at 4°C.

The schematic diagram of the structure of the affinity column prepared in Example 1 is shown in FIG.

the preparation and working principle of the aptamer affinity column is shown in FIG. 2, and the test

esults of the number of repeated uses and the recovery rate are shown in FIG. 3. The aptamer affinity

:olumn of Alternariol of the invention can be reused more than 20 times, and the average recovery rate is

nore than 85%.

Example 2 Purification of Alternariol in lotus seed samples and its detection using an aptamer

ffinity column of Alternariol

In this example, a standard substance of Alternariol was quantitatively added to a lotus seed sample,

allowed by purification with the Alternariol aptamer affinity column prepared in Example 1. After

>urification, a high performance liquid chromatography-fluorescence detector was used to determine the

ecovery rate. The details were given below:

1. Lotus seed sample processing

1) the lotus seed sample was crushed.

2) According to the standards of 0.5 ng, 5 ng, and 50 ng per gram of the sample, the Alternariol

tandard was added to the crushed lotus seed sample.

3) 25 mL methanol-water (7:3, v/v) was added to 5 g of sample, and placedin a multi-tube shaker

or high-speed homogenization at 2500 rpm for 20 min.

4) 45mL binding buffer was add to 5mL supernatant, vortexed, centrifuged at 10000rpm for 10min,

and filtered with glass fiber filter paper to collect the filtrate.

5) 20 mL of the above-mentioned filtrate was taken for sample purification and testing.

2. the aptamer affinity column of Alternariol was taken out, the injection port plug was opened, and

the affinity column was equilibrated with 5 mL binding buffer (10 mM Tris, 120 mM NaCl, 5 mM KCl

and 5 mM MgC2, pH 7.5). 20 mL of the above-mentioned filtrate was added to the affinity column, and

the injection port was connected to the syringe barrel to allow the liquid to flow out at a flow rate of 1-2

drops per second until all the samples flow out of the affinity column.

3. the affinity column was washed with 1 mL Binding Buffer, and 2 to 3 mL of air was injected into

the column.

4. after addition of 1 mL of methanol, the eluted products were collected, and 2 to 3 mL of air was injected into the column. 5. 1 mL of methanol was added again, and the eluted products were not collected. 6. the eluted products collected above was purged with nitrogen in a nitrogen blowing instrument at

[0C, then it was redissolved with 1mL methanol-water (55:45, v/v), and filtered with a 0.22tm filter ilm for use of HPLC- Fluorescence detector (HPLC-FLD) detection. 7. the detection results of high performance liquid chromatography was shown in Table 1. Table 1 HPLC-FLD test results of the content of Alternariol in lotus seeds Standard amount Alternariol Recovery rate 0.5ng/g AOH 85.73 5ng/g AOH 109.56 50ng/g AOH 106.03 It can be seen that after the lotus seed sample was purified by the Alternariol aptamer affinity column, Uiternariol can be detected by HPLC-FLD. It can be seen from the test results that when 0.5 ng, 5 ng, and ng of Alternariol were added to 1 g of the crushed lotus seed sample, the AOH recovery rate of the ptamer affinity column of the present invention was between 85.73% and 109.56%. Example 3 Design and screening of Alternariol specific DNA aptamers Aiming at the problems of few binding sites of small molecule targets and nucleic acids, difficult eparation and fixation, and a great influence on their chemical bonds after fixation which will lead to creening failures, this Example used a fixed library to replace the traditional method offixing small nolecules and established a suitable screening method for small molecule compounds such as AOH, vhich solved the difficult problem that small molecule targets are difficult to fix and separate. The main >rinciple was to pair the biotin-modified capture nucleic acid with the annealing of the library. Through the interaction of biotin and SA, the library was connected to the SA magnetic beads and incubated with the target. The specifically bound aptamer will be competitively eluted, followed by collecting the bound amplified nucleic acid to prepare a single strand for the next round of screening. 1. Design of library: '-ATTGGCACTCCACGCATAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNCCTATGCGTGCTACCGTGAA-3' The secondary structure of the library was shown in Figure 4. 2. Library fixation and screening: (1) 100ul secondary library was added into PCR tube, 1.4uL liblOSICS (100uM, DPBS dissolved) was added, centrifuged and mixed.

(2) slow denaturation and renaturation were performed in a PCR instrument. The procedures for

etting the PCR instrument was: 95°C for 10 minutes, cooling to 60°C at 0.1°C/sec, keeping for 1 minute,

nd then cooling to 25°C at 0.1°C/sec to obtain a sample pool.

(3) 70ul SA beads were washed 5 times with DPBS, 200ul each time, and a magnet was used to fish

he beads and the supernatant was removed.

(4) the pool in step (2) was added to SA beads, and incubated for 0.5h in a shaker at room

emperature.

(5) a magnet was used to fish the beads and the supernatant was removed, followed by washing with

)PBS (98ul DPBS+2uL methanol) 6 times, 200ul each time. a magnet was used to fish the beads and the

upernatant was removed. The supernatant was marked as wash, wash2, wash3, wash4, wash5 and

vash6.

(6) a small amount of beads was taked for check of the quality of the library with a flow cytometer.

[he results were shown in Figure 5.

(7) the change of pool concentration was measured before and after adding beads with a fluorescence

pectrometer, and the dissociation of the library from the beads during quantitative rinsing with Q-PCR

vas show in Table 2.

Table 2 Fluorescence spectrometer measures the effect of library fixation

Sample

pool before adding beads 81.6

pool after adding beads 8.0

Percentage of pool fixed with beads 90%

(8) 100ul AOH solution (2uL AOH was added to 98ul DPBS, the final concentration of AOH was

1OOuM) was added to SA beads, mixed well and incubated for lh in a shaker at room temperature. The

beads were fished with a magnet, and the supernatant was recovered as elution 1.

(9) Q-PCR detection. lul sample was added to 40ul mix. The Q-PCR results of single-stranded

amplification were shown in Figure 6.

(10) the binding nucleic acids were collected and amplified to prepare a single strand for the next

round of screening. The final aptamer was obtained through 6 rounds of screening and sequenced (SEQ

ID NO: 1).

3. Determination of affinity and specificity of aptamers obtained by screening

Fluorescence method was used to determine the affinity. Because AOH had an excitation wavelength, the affmity of the aptamers and the target toxin can be determined by detecting fluorescence. If the >henomenon of increased fluorescence was found, it can indicate that AOH binds to DNA. (1) The final concentration of AOH is luM, and the single clone was diluted and sampled according o the final concentration in the table below. The volume of each sample was OOuL, and the dilution >uffer was DPBS. Three repeats were made for each sample.

control clone 15nM aptamerl 15nM aptamer2 15nM aptamer3 15nM control clone 31nM aptamerl 31nM aptamer2 31nM aptamer3 31nM control clone 62nM aptamerl 62nM aptamer2 62nM aptamer3 62nM control clone 125nM aptamerl 125nM aptamer2 125nM aptamer3 125nM control clone 250nM aptamerl 250nM aptamer2 250nM aptamer3 250nM control clone 500nM aptamerl 500nM aptamer2 500nM aptamer3 500n control clone 10OOnM aptamerl 1000nM aptamer2 1OOnM aptamer3 1000n4

(2) The microplate reader was used to scan, with excitation at 350nm, it was found that the luorescence emitted at 440nm, and the data obtained from three replicate wells were averaged. The obtainedd AOH aptamer affinity determination results were shown in Figure 7. (3) The specificity of the aptamer was determined by the fluorescence method with the other five oxins as interferents. See Figure 8 for the aptamer-specific detection results. For AOH and its structural analogue Alternariol monomethyl ether AME, there is only one methyl difference in chemical structure (Figure 9), but the aptamers obtained by screening have a significant difference in affinity for the two compounds, which proves the aptamers screened in the present invention lave very good specificity (Figure 10). Although the general description and specific embodiments have been used to describe the present invention in detail above, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

Claims (10)

What is claimed is:

1. An Alternariol-specific DNA aptamer, wherein the nucleic acid sequence is as shown in SEQ ID

:1.

2. A detection reagent, a detection test strip, a detection kit or an affinity column comprising the

ptamer of claim 1.

3. Use of any one of the aptamer of claim 1:

(1) use in the preparation of aptamer affinity column of Alternariol;

(2) use in the preparation of test reagent, test strip or test kit for Alternariol;

(3) use in the detection of Alternariol.

4. The aptamer affinity column of Alternariol, wherein the packing of the affinity column is obtained

>y using NHS-activated sepharose as a carrier and covalently coupling the carrier with the aptamer

ccording to claim 1.

5. The affinity column according to claim 4, wherein the aptamer is a chemically modified aptamer

equence, and the modification method includes amino modification, carboxyl modification, sulfhydryl

nodification or biotin modification.

6. The affinity column according to claim 5, wherein the aptamer is an amino-modified aptamer

equence, and the modification method is given below: the 3' or 5' end of the nucleic acid aptamer is

:ovalently bonded to C7 indirect arm -(CH 2 ) 7 - or C6 indirect arm -(CH 2 ) 6-, and then the end of C7

ndirect arm or C6 indirect arm is modified with amino group through covalent bond to obtain an

Imino-modified aptamer.

7. A method for preparing the affinity column according to claim 6, comprising the following steps:

1) Preparation of NHS-activated sepharose

( 10-15 mL sepharose gel 4FF is washed with water and vacuum filtered, washed with 30-50% and

-70% acetone in a funnel, and finally washed with dioxane several times, vacuum filtered and

transferred to a triangular flask, and then 10-15mL dioxane, 100-300pL allyl glycidyl ether and

300-1000L boron trifluoride ethyl ether, and shaked at 120-180 rpm and 30-40°C for 45-60 min in a

shaker; the resulting matrix after reaction is washed with 30-50% and 50-70% acetone in sequence, and

finally washed with deionized water to obtain an activated matrix;

@carboxylation of thioglycolic acid: 1.20-2mL thioglycolic acid, 10-15mL deionized water and

0.25-0.5g ammonium persulfate are added to 10-15mL activated matrix, and reacted at 60-80°C for 8-12

h to obtain a thioglycolic acid carboxylated sepharose gel;

@ coupling of NHS groups: 10-15 mL thioglycolic acid carboxylated sepharose gel is washed with

-50%, 70-90% and 100% acetone solution, and then washed with dioxane several times to obtain a

ioxane-modified sepharose gel; the dioxane-modified sepharose gel is transferred to a triangular flask,

ind 10-15mL dioxane, 1-5g NHS and 1-5g N,N-dicyclohexylcarbimide are added, shaked and reacted at

!5-30°C for 8-12h; finally, the matrix is washed with dioxane, methanol, and acetone in sequence to

>btain a NHS-activated sepharose that will be stored in isopropanol away from light.

2) Preparation of amino-modified aptamer of Alternariol

the 3' end of the nucleic acid aptamer is covalently bonded to C7 indirect arm -(CH 2) 7-, and then the

nd of the C7 indirect arm is covalently modified with an amino group to obtain an amino-modified

ptamer;

3) Washing of NHS-activated sepharose: 300-500 pL the NHS-activated sepharose was added in a

entrifuge tube and washed with 1-1.5 mM hydrochloric acid several times, 1-2 mL each time to obtain a

washed carrier ;

4) Renaturation of the aptamer: 1-5 OD amino-modified aptamer of Alternariol is dissolved in

!00-1000 pL Na 2HPO4 buffer, renaturated at 75-95°C for 3-5 min, and then placed at room temperature

or 15-60 min to obtain a renaturated aptamer solution; wherein the Na2HPO 4 buffer is: 200 mM

4a 2 HPO4, 5mM MgC2, pH 8.0;

5) Coupling: 200-1000 L renaturated aptamer solution in step 4) is added to the washed carrier in

tep 3), and shaked overnight at 25-35°C in a shaker;

6) Blocking: After centrifugation of the obtained coupling product in Step 5) to remove the

supernatant, 1-5 mL blocking buffer is added, and shaked at 30-40°C for 2-5 hours in a shaker to block

the remaining active sites to obtain a carrier-aptamer coupling gel; wherein the blocking buffer is: 0.2%

BSA, 0.1M MES, 0.15M NaCl, pH 6.0;

7) Washing: the above-mentioned carrier-aptamer coupling gel is washed with washing buffer

several times to remove uncoupled aptamers; the washed coupling gel is resuspend with 1-5 mL of

binding buffer, and the resulting coupling gel suspension is ready to be packed into the column; wherein

the washing buffer is: 50mM Tris-HCl, 0.15M NaCl, pH 7.2; the binding buffer is: 10 mM Tris HCl, 120

mM NaCl, 5 mM KCl, 1 mM MgC2;

8) Packing the column: a solid phase extraction column with a volume of 1-5 mL is padded well with

a lower sieve plate, and the column is packed with the above coupling gel suspension until the gel height

is 1-2 cm, and 0.5-3 mL of 0.02-0.05% w/v NaN3 solution is added, and stored at 4-10°C.

8. The method according to claim 7, wherein the material of the solid phase extraction column and

he lower sieve plate in step 8) is selected from the group consisting of polypropylene, polystyrene,

>orous polystyrene or cross-linked porous polystyrene; and /or

the aperture size of the lower sieve plate is 10 m.

9. Use of the affinity column according to any one of claims 4-6:

9use in the enrichment and purification of Alternariol in samples;

O use in the purification of Alternariol in samples.

10. The use according to claim 9, wherein the samples include food, feed, grain crops and traditional

Phinese medicine.