CN111662900A

CN111662900A - Sulfamethazine aptamer screening method, kit and application

Info

Publication number: CN111662900A
Application number: CN202010401091.5A
Authority: CN
Inventors: 乐涛; 寇启明; 孙琦; 吴平
Original assignee: Chongqing Normal University
Current assignee: Chongqing Normal University
Priority date: 2020-05-13
Filing date: 2020-05-13
Publication date: 2020-09-15
Anticipated expiration: 2040-05-13
Also published as: CN111662900B

Abstract

The invention provides a screening method of sulfadimidine aptamer and a detection kit thereof, wherein the kit comprises a graphene oxide-based fluorescent aptamer sensor; the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table. Application of the sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a test sample for detecting milk or eggs.

Description

Sulfamethazine aptamer screening method, kit and application

Technical Field

The invention relates to the field of biotechnology and food antibiotic residue detection. More specifically relates to a screening method, a kit and application of a sulfadimidine aptamer.

Background

Sulfamethazine, chemically known as 2- (p-aminophenylsulfonamide) -4, 6-dimethylpyrimidine, is a common sulfonamide drug, is a broad-spectrum antibacterial agent, can be used for clinical treatment, can also be used as a veterinary drug and a feed additive, is widely applied to feeding of food-borne animals, and can be transferred to animal-borne foods such as meat, eggs, milk and the like after entering the animal body through various administration routes. If the food is improperly used, residues can be easily generated in animal tissues and animal-derived foods and enter human bodies through food chains, so that the human health is harmed. And is discharged into the external environment through animal dung, urine and other ways, thus causing ecological toxicological pollution. In order to ensure the safety of animal-derived foods, the Maximum Residual Limit (MRL) of sulfonamides such as sulfadimidine in animal-derived foods is regulated by governments of various countries. The total amount of the sulfonamides in food and feed is regulated by the international food code commission (CAC), the World Health Organization (WHO) and the FDA (food and drug administration) in the United states to be not more than 0.1mg/kg, the maximum residual limit of the sulfonamides in animal-derived food in China is 100ng/mL, and the total amount of various sulfonamides is not more than 100 ng/mL. Therefore, a rapid, efficient and accurate sulfadimidine detection method is established, and the method has very important significance for ensuring the dietary health of human beings and reducing ecological pollution. At present, methods such as high performance liquid chromatography, chromatography-mass spectrometry combined technology, enzyme-linked immunosorbent assay and the like are mostly adopted for detecting the sulfadimidine drug, and although the methods have high selectivity and sensitivity, the use of the chromatography requires relatively expensive analytical instruments and professional technicians, and particularly has the defects of complicated sample pretreatment process, low extraction efficiency of low-content target substances, high sample detection cost, matrix interference, difficulty in realizing rapid detection and the like. The enzyme-linked immunosorbent assay needs to prepare antibodies, while the preparation of drug antibodies needs to synthesize complete antigens and immunize animals, the experimental period is long, and the biological activity is easily inactivated due to the influence of various factors. In recent years, the biosensor method for detecting the drugs is more and more concerned by researchers due to the advantages of simple operation, high detection speed, low price and the like. The biosensor constructed based on the aptamer has the advantages of high detection sensitivity, simplicity in operation and strong selectivity, and has a good application prospect in antibiotic drug detection. According to the invention, the sulfadimidine aptamer is screened and sequence optimized based on the graphene oxide, the fluorescent aptamer sensor based on the graphene oxide is constructed, the fluorescent aptamer sensor is successfully detected in actual samples of milk and eggs, the linear relation is good, and a foundation is laid for the detection and development of sulfadimidine products.

The aptamer is a single-stranded DNA or RNA fragment which is screened out in vitro by Exponential Enrichment of ligand by expression evolution (SELEX) and can be combined with various target molecules with high affinity and high specificity, can form a secondary or tertiary structure through self folding to ensure that the aptamer has strong affinity to specific targets (such as metal ions, small molecules, proteins, viruses, cells and the like), and is used as an element for molecular recognition, thereby being widely applied to medical clinical diagnosis and treatment.

At present, the report of applying the aptamer as a probe to the detection of the antibiotic detection residue in food is available, the detection method mainly comprises an immunoassay method and an electrochemical biosensor, and the detection of the sulfadimidine residue in food and environment must be enhanced in order to ensure the quality of animal-derived food and guarantee the food safety of human beings.

The invention content is as follows:

according to the invention, through a screening process of a non-fixed GO-SELEX technology, a nucleic acid aptamer of sulfadimidine is obtained, an aptamer core recognition region is obtained through sequence optimization, and is cloned and synthesized in vitro, the prepared nucleic acid aptamer has high affinity and specificity to sulfadimidine, a graphene oxide-based fluorescent aptamer sensor is constructed, and when sulfadimidine appears in a system, the nucleic acid aptamer is combined to the sulfadimidine through strong affinity and is not adsorbed on the surface of the graphene oxide through pi-pi accumulation. Due to the difference of sulfadimidine concentration, the combined fluorophore-labeled aptamer generates different fluorescence signals. In contrast, in the absence of sulfadimidine, graphene oxide can adsorb fluorophore-labeled aptamers through pi-pi stacking interactions, quenching the fluorescent signal. By utilizing the principle, the quantitative detection of the sulfadimidine is realized. The specific invention content is as follows:

the sulfadimidine fluorescence detection kit comprises a graphene oxide-based fluorescence aptamer sensor; the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the nucleic acid adapter sequence of the sulfadimidine marked by the FAM fluorescent group is one of No.1, No.2, No.3, No.4, No. 5, No.6, No1s or No 5s recorded in a nucleotide sequence table. Aptamer derivatives obtained by modifying and modifying ssDNA, and the like, also belong to the protection scope of the invention.

The sulfamethazine nucleic acid adaptor sequence marked by the FAM fluorescent group is one of No1s or No 5s recorded in a nucleotide sequence table.

The sulfamethazine nucleic acid adaptor sequence marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table.

1) Screening a sulfadimidine aptamer by adopting a non-immobilized GO-SELEX technology;

2) obtaining an aptamer core recognition region through sequence optimization, and cloning and synthesizing in vitro to prepare the aptamer with high affinity and specificity to the sulfadimidine;

a method for screening a sulfadimidine aptamer, comprising the steps of:

2) an aptamer core recognition region is obtained through sequence optimization, and is cloned and synthesized in vitro to prepare the aptamer with high affinity and specificity to the sulfadimidine.

The construction method of the graphene oxide-based fluorescence aptamer sensor comprises the following steps: the prepared aptamer with high affinity and specificity to sulfadimidine is used for constructing the graphene oxide-based fluorescent aptamer sensor.

Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a milk test sample.

Application of a sulfadimidine fluorescence detection kit in qualitative detection of sulfadimidine in an egg tissue test sample.

The beneficial technical effects of the invention are as follows: aiming at the defects that the traditional large instrument can not realize on-site rapid detection and is complex to operate, and the immunoassay method needs to prepare antibodies through experimental animals and has long periodicity, the invention establishes the graphene oxide aptamer sensor-based fluorescence detection method, can be used for rapid and high-sensitivity quantitative detection of sulfadimidine residues in food, and overcomes the defects of the detection method. Provides a new method for detecting antibiotics. The method has the advantages of simple detection operation, non-fixed target, short screening rounds and the like, and lays a foundation for food safety detection and product development.

Drawings

FIG. 1: (ii) aptamer affinity assay;

FIG. 2: specific analysis of fluorescent aptamer sensors;

FIG. 3: fluorescence calibration curves plotted at emission wavelength 520nm using different concentrations of standards.

Detailed Description

Example 1

1. GO-SELEX process for screening aptamers to sulfadimidine

1) Performing library renaturation treatment: a500 nM library (5'-FAM-GACAGGCA GGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3'; N is a random sequence) is placed in a metal bath and heated at 95 ℃ for 10min, then rapidly placed on ice and placed for 10min, and then taken out and kept in the dark for balancing at room temperature for 25min, so that a large amount of ssDNA in the library is folded to form a complex and diversified three-dimensional structure. And the fluorescence value was measured.

2) First screening: adding 7 mu L of sulfadimidine solution (4 mu g/mL) into 200uL Lib, placing the mixture on a shaker at 25 ℃ and 250rpm for incubation for 1h in the dark, wherein ssDNA is self-adaptive to form a three-dimensional structure, and the three-dimensional structure formed by a part of ssDNA can be combined with sulfadimidine to form a ssDNA-sulfadimidine compound. Adding a certain proportion of GO, placing on a shaking table at 25 ℃, and incubating for 20min in a dark place at 250rpm, wherein free ssDNA is uniformly adsorbed on graphene oxide through pi-pi accumulation, and the ssDNA-sulfadimidine compound cannot be bound on the graphene oxide. Then, the mixture was centrifuged at 15000rpm at 25 ℃ for 10min, the precipitate was discarded, and the supernatant was recovered. The supernatant contained ssDNA capable of binding to sulfadimidine, fluorescence intensity (492nm excitation, 520nm emission) was measured on a microplate reader, and the recovery rate was calculated.

Taking supernatant collected after incubation in a proper amount as a template, carrying out PCR amplification by using a forward primer with a fluorescent label and a backward primer with biotin according to the reaction conditions, and carrying out polyacrylamide gel electrophoresis verification.

3) And (3) calculating the recovery rate: recovery was calculated after each screening incubation and calculated as (F)_{Recovered ssDNA}/F_{Input ssDNA}) × 100%, along with the screening, ssDNA which can not be combined with sulfadimidine is gradually screened, ssDNA which can have high affinity with sulfadimidine is continuously enriched, the recovery rate is continuously increased until the recovery rate is stable, the affinity aptamer of sulfadimidine is enriched, and the screening process is basically finished.

4) Negative screening: and when the recovery rate tends to be stable, carrying out one round of negative screening. And (3) respectively adding sulfamquinoxaline, sulfamdimethoxypyrimidine and sulfamethoxydiazine which are equivalent to the library in molar quantity after the secondary library prepared in the previous round is subjected to renaturation treatment, incubating for 1h in a dark place on a shaking table at 25 ℃ and 250rpm, adding graphene oxide with the mass ratio of the library to the graphene oxide being 1:24, incubating for 20min in a dark place on a shaking table at 25 ℃ and 250rpm, dissociating ssDNA (single-stranded deoxyribonucleic acid) which can be combined with the sulfamquinoxaline, the sulfamethoxydiazine and the sulfamethoxydiazine in a liquid, adsorbing ssDNA (single-stranded deoxyribonucleic acid) which can not be combined with the ssDNA in a liquid, centrifuging for 10min at 15000rpm and 25 ℃, and discarding a supernatant. Adding a binding buffer solution into a centrifugal tube containing the graphene oxide precipitate, centrifuging to remove supernatant, and repeating the operation for 3 times to completely remove ssDNA capable of binding with sulfaquinoxaline, sulfadimethoxine and sulfadimethoxine. Adding 200 mu L of binding buffer solution, adding equimolar sulfadimidine, incubating for 1h at 25 ℃ and 250rpm in a shaking table in the dark, desorbing the compound formed by ssDNA specifically bound with the sulfadimidine from graphene oxide, centrifuging to obtain supernatant, and measuring the fluorescence intensity by using a microplate reader.

5) And (4) final wheel screening: and repeating the positive screening process of the ssDNA obtained after the negative screening, calculating the recovery rate, and then performing one round of positive screening to enrich the sulfadimidine aptamer.

2. Preparation of the Secondary library

1) And (3) cleaning magnetic beads: taking 2 tubes of 600 mu LPromega magnetic beads, 1 XPBS (0.1mg/mLBSA), washing for 3 times at 1 mL/time;

2) resuspending magnetic beads: resuspend with 800. mu.L of 1 XPBS (0.1mg/mLBSA, 0.2M NaCl, pH 7.4), respectively;

3) and (3) extracting dsDNA: adding the PCR products into 800 mu L of magnetic bead suspension respectively, shaking for 1h at 25 ℃ and 280rpm in the dark, and connecting the DNA to streptavidin magnetic beads through the action of biotin and streptavidin;

4) isolation of dsDNA: separating with magnetic frame to remove supernatant, washing with 1mL of 1 × PBS (pH 7.4) for 3 times to remove DNA not bound to the magnetic beads, and combining two tubes of magnetic beads to remove supernatant;

5) ssDNA regeneration: adding 50 μ L of 0.05M NaOH, vortex reacting for 2min to separate the sense strand and the antisense strand under alkaline condition, attaching the ssDNA strand with biotin to the magnetic bead, cleaving the other strand without biotin, and adding 25 μ L of ddH₂O and 100. mu.L of 2 × Tris-HCl continue to swirl and oscillate for 1min, the supernatant is carefully collected by magnetic separation, 25. mu.L of 0.1M HCl is added into the collected liquid to neutralize NaOH, 200. mu.L of secondary library is obtained, finally, the fluorescence intensity (492nm excitation and 520nm emission) is measured by a microplate reader, the secondary library concentration is repeatedly measured for 3 times, and the secondary library is calculated according to a standard curve.

3. Cloning and sequencing

ssDNA obtained from 7 rounds of screening was PCR amplified using unlabeled forward and backward primers, and clones were selected for sequencing. The sequencing results are shown in table 1.

TABLE 1

4. Nucleic acid sequence analysis and optimization

Firstly, sequence comparison and homology analysis are carried out on a sequence obtained by sequencing by using software DNAMAN, and the characteristics of each nucleic acid sequence are analyzed; then, predicting the secondary structure of the nucleic acid sequence on line by using MFold; and (3) carrying out affinity determination on No1 and No 5, and then appropriately truncating the nucleic acid sequence according to the secondary structure to improve the affinity, thereby obtaining two tailored aptamers No1s and No 5s, which are specifically shown in Table 2.

TABLE 2

No1s	CGTTAGACG
		No5s	GCTGATAGC

5. Affinity assay

The graphene oxide can adsorb ssDNA, has a good fluorescence quenching effect, and can quench a fluorescent group. Gradient concentrations (12.5, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0nmol/L) of 5' -FAM fluorescently labeled candidate aptamers were mixed with equimolar amounts of sulfadimidine, respectively, and incubated at 25 ℃ for 1h in the dark. Then, adding graphene oxide solution according to different ssDNA/GO mass ratios, fully mixing, and continuously incubating for 20min at 25 ℃ in the dark. The supernatant containing the sulfadimidine-aptamer complex is absorbed after centrifugation for 10min at 15000rpm and 25 ℃, and the fluorescence intensity (excitation at 492nm and emission at 520nm) is measured by a microplate reader. At the same time, candidate aptamer sequences at each concentration were incubated separately in mixtures with different mass ratios of GO (no target added)Standard sulfadimidine) as a negative control, eliminating the effect of candidate aptamer sequences from self-desorption from GO surfaces. Nonlinear regression analysis was performed on each sequence using GraphPad Prism 5.0 software, a binding saturation curve was plotted using the candidate aptamer concentration as the abscissa and the difference in fluorescence intensity (Δ F) between the experimental group and the control group as the ordinate, and the dissociation constant (K) was calculated_d). As shown in fig. 1, the affinities of No1, No 5, No1s, No 5s are: 167.8nM, 185.7nM, 76.4nM, 81.3 nM.

The aptamer derivatives obtained by modifying and modifying the DNA fragments also belong to the protection scope of the invention.

6. Experiment on binding specificity of aptamer and sulfadimidine

Taking sulfonamides of the same type as sulfadimidine: the specificity of the aptamer obtained by screening is measured by using sulfadimethoxine, sulfamethoxydiazine, sulfaquinoxaline, different antibiotics nitrofuran and climbazole. Respectively carrying out binding reaction on 100nmol/L aptamer and sulfamethoxydiazine, sulfaquinoxaline, sulfadimethoxine, nitrofuran and climbazole for 60min, adding graphene oxide with the same mass ratio, slightly shaking and incubating for 20min, measuring the fluorescence intensity of a supernatant by using an enzyme labeling instrument, and analyzing the specificity of the aptamer through the fluorescence intensity (emission wavelength of 520nm) of a candidate drug. As shown in FIG. 2, the fluorescence intensity of sulfadimidine was the highest compared to the other antibiotics, indicating that the No1s aptamer has high specificity for sulfadimidine.

7. Standard curve establishment

FAM fluorescently labeled aptamer (100nM) was incubated with a series of sulfadimidine concentrations of 0.4-500ng/mL in 200 μ L of binding buffer in the dark at 25 ℃ for 1h, GO was added to the mixture, incubation in the dark at room temperature for 20min, fluorescence intensity was measured by a microplate reader at 520nM emission wavelength and a standard curve was plotted. FIG. 3 is a graph showing the relationship between the fluorescence intensity of sulfadimidine (0.4 ng/mL-500 ng/mL) and the No1s aptamer at different concentrations.

EXAMPLE 2 determination of Sulfamethazine in real samples

Samples of milk and eggs purchased from local supermarkets, previously analyzed by HPLC, proved to be free of SMZ compounds. A10 ml sample of milk was taken and first centrifuged at 14,000 rpm at 4 ℃ for 20 minutes. The supernatant was diluted to 100mL with binding buffer and filtered through a 0.22- μm microfiltration membrane. For egg samples, after thorough mixing and homogenization, 2g of egg sample was added to the centrifuge tube. Then 4mL ethyl acetate was added and shaken for 3 min. After centrifugation at 5000 Xg for 5min at room temperature, the supernatant was dried in a water bath at 80 ℃ or in nitrogen and diluted to 500. mu.L with binding buffer. To evaluate the recovery of the established graphene oxide-based fluorescent aptamer sensor, samples were added with different concentrations of sulfadimidine (2.0, 10.0, 25.0, 50.0, and 100.0ng/mL), respectively, and detection was performed with the established aptamer sensor. And drawing a curve according to the operation method of the step seven, and carrying out experiment and result analysis.

The lowest limit of detection (LOD) is calculated as: LOD 3 SD/slope, where SD represents the standard deviation of fluorescence values measured for 20 blank samples (milk and egg), respectively. And substituting the equation to calculate the detection limit of the sample. The experimental results are as follows: in the test of sulfadimidine in milk and egg samples, the detection limit of the samples is 1.37 mug/kg and 3.15 mug/kg respectively.

The recovery rate is shown in the table 3, the recovery rate of the milk sample is between 94.4% and 108.8%, and the recovery rate of the egg sample is between 93.9% and 106.7%. The method respectively detects the variation coefficient ranges in milk and eggs as follows: 5.4 to 12.7, 5.1 to 8.8. According to the results of the coefficient of variation and the recovery rate, the method has better repeatability and accuracy and can meet the requirement of on-site detection of food and medicine. See table 3 for details.

Table 3 accuracy and precision of sulfadimidine addition to eggs and milk (n ═ 5).

Sequence listing

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Claims

1. A screening method of a sulfadimidine aptamer is characterized in that: the method comprises the following steps:

2. The sulfadimidine fluorescence detection kit comprises a graphene oxide-based fluorescence aptamer sensor; the method is characterized in that: the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No.1, No.2, No.3, No.4, No. 5, No.6, No1s or No 5s described in a nucleotide sequence table.

3. The sulfadimidine fluorescence detection kit of claim 2, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No.1, No 5, No1s or No 5s recorded in a nucleotide sequence table.

4. The sulfadimidine fluorescence detection kit of claim 3, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No1s or No 5s recorded in a nucleotide sequence table.

5. The sulfadimidine fluorescence detection kit of claim 4, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table.

6. The sulfadimidine fluorescence detection kit of claim 2, characterized in that: the fluorescent aptamer sensor based on graphene oxide is constructed on the basis of the aptamer with high affinity and specificity of sulfadimidine obtained by the method of claim 1.

7. Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a milk test sample.

8. Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in an egg tissue test sample.