CN111662900A - Sulfamethazine aptamer screening method, kit and application - Google Patents

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

A kind of sulfamethazine nucleic acid aptamer screening method, kit and application

technical field

The invention relates to the fields of biotechnology and food antibiotic residue detection. More specifically, it relates to a screening method, kit and application of sulfamethazine nucleic acid aptamer.

Background technique

Sulfamethazine, the chemical name is 2-(p-aminobenzenesulfonamido)-4,6-dimethylpyrimidine, is a common sulfonamide drug and a broad-spectrum antibacterial agent. It can also be used as a veterinary drug and feed additive, and is widely used in the feeding of food-derived animals. After entering the animal through various routes of administration, it can be transferred to animal-derived foods such as meat, eggs, and milk. If used improperly, it is easy to produce residues in animal tissues and animal-derived foods, and enter the human body through the food chain, causing harm to human health. And it is discharged into the external environment through animal feces and urine, resulting in ecotoxicological pollution. In order to ensure the safety of animal-derived food, governments of various countries have stipulated the maximum residue limit (maximum residue limit, MRL) of sulfonamides such as sulfamethazine in animal-derived food. The International Codex Alimentarius Commission (CAC), the World Health Organization (WHO) and the US FDA (Food and Drug Administration) stipulate that the total amount of sulfonamides in food and feed shall not exceed 0.1 mg/kg. The maximum residue limit in food is 100ng/mL, and the total amount of various sulfonamides shall not exceed 100ng/mL. Therefore, the establishment of a fast, efficient and accurate detection method for sulfamethazine is of great significance to ensure the health of human diet and reduce ecological pollution. At present, the detection of sulfamethazine drugs mostly adopts high performance liquid chromatography, chromatography-mass spectrometry and enzyme-linked immunosorbent assay. Although these methods have high selectivity and sensitivity, chromatography Its use requires relatively expensive analytical instruments and professional technicians, especially the disadvantages of cumbersome sample pretreatment process, low extraction efficiency for low-content target compounds, high sample detection cost, matrix interference, and difficulty in achieving rapid detection. The enzyme-linked immunosorbent assay requires the preparation of antibodies, while the preparation of drug antibodies requires the synthesis of complete antigens and immunization of animals. The experimental period is long, and the biological activity is easily inactivated by various factors. In recent years, biosensor method for drug detection has attracted more and more attention of scientific researchers due to its advantages of simple operation, fast detection speed and low price. Biosensors based on nucleic acid aptamers have high detection sensitivity, simple operation and strong selectivity, and have good application prospects in antibiotic drug detection. In the present invention, the sulfamethazine nucleic acid aptamer is screened and sequence optimized based on graphene oxide, and a graphene oxide-based fluorescent aptamer sensor is constructed, which is successfully detected in actual samples of milk and eggs, and has a good linear relationship. The foundation has been laid for the detection and development of sulfamethazine.

Nucleic acid aptamers are a kind of single-stranded DNA or RNA that can bind to various target molecules with high affinity and high specificity, which is screened by the exponential enrichment of ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX) in vitro. Fragments, which can form secondary or tertiary structures through self-folding, make them have a strong affinity for specific targets (such as metal ions, small molecules, proteins, viruses, cells, etc.), and are widely used as molecular recognition elements. Clinical diagnosis and treatment.

At present, there have been reports on the use of nucleic acid aptamers as probes for the detection of antibiotic residues in food. The detection methods are mostly immunoassays and electrochemical biosensors. Safety, the detection of sulfamethazine residues in food and the environment must be strengthened.

Invention content:

The invention obtains the nucleic acid aptamer of sulfamethazine through the screening process of the non-fixed GO-SELEX technology, obtains the core recognition region of the aptamer through sequence optimization, and clones the nucleic acid aptamer synthesized in vitro. With high affinity and specificity for sulfamethazine, a graphene oxide-based fluorescent aptamer sensor was constructed. When sulfamethazine appears in the system, the nucleic acid aptamer binds to sulfamethazine with strong affinity on the surface of graphene oxide without π-π stacking. The conjugated fluorophore-labeled aptamer produces different fluorescent signals due to different sulfamethazine concentrations. In contrast, in the absence of sulfamethazine, graphene oxide can adsorb fluorophore-labeled aptamers via π-π stacking interactions, quenching the fluorescence signal. Using this principle, the quantitative detection of sulfamethazine was realized. The specific contents of the invention are:

A sulfamethazine fluorescence detection kit, comprising a graphene oxide-based fluorescent aptamer sensor; wherein the graphene oxide-based fluorescent aptamer sensor includes an aptamer labeled with a FAM fluorophore and graphene oxide; the The sulfamethazine nucleic acid adapter sequence labeled with FAM fluorophore is No.1, No.2, No.3, No.4, No.5, No.6, No.1s or No.5s described in the nucleotide sequence table. one. Nucleic acid aptamer derivatives obtained by modifying and transforming the above-mentioned ssDNA also belong to the protection scope of the present invention.

The FAM fluorescent group-labeled sulfamethazine nucleic acid adaptor sequence is one of No1s or No5s recorded in the nucleotide sequence table.

The FAM fluorescent group-labeled sulfamethazine nucleic acid adaptor sequence is No1s recorded in the nucleotide sequence table.

1) Screening of sulfamethazine aptamers using non-fixed GO-SELEX technology;

2) Through the optimization of the sequence, the core recognition region of the aptamer is obtained, and the clone is synthesized in vitro to obtain the nucleic acid aptamer with high affinity and specificity to sulfamethazine;

A screening method for sulfamethazine nucleic acid aptamer, comprising the following steps:

1) Screening sulfamethazine aptamers by non-fixed GO-SELEX technology;

2) Through the optimization of the sequence, the core recognition region of the aptamer is obtained, and the clone is synthesized in vitro to obtain a nucleic acid aptamer with high affinity and specificity to sulfamethazine.

The construction method of the graphene oxide-based fluorescent aptamer sensor is as follows: the graphene oxide-based fluorescent aptamer sensor is constructed by using the prepared nucleic acid aptamer with high affinity and specificity to sulfamethazine.

Application of sulfamethazine fluorescence detection kit in the quantitative detection of sulfamethazine in milk test samples.

Application of sulfamethazine fluorescence detection kit in the qualitative detection of sulfamethazine in egg tissue test samples.

The beneficial technical effects of the present invention are as follows: in view of the shortcomings that traditional large-scale instruments cannot achieve rapid on-site detection, cumbersome operations, and the immunoassay method needs to prepare antibodies through experimental animals with long periodicity, a graphene oxide aptamer sensor fluorescence sensor is established. The detection method can be used for rapid and high-sensitivity quantitative detection of sulfamethazine residues in food, and overcomes the defects of the above detection methods. Provides a new method for antibiotic detection. It has the advantages of simple detection operation, non-fixed target and short number of screening rounds, which lays the foundation for food safety detection and product development.

Description of drawings

Figure 1: Nucleic acid aptamer affinity determination;

Figure 2: Specificity analysis of fluorescent aptamer sensors;

Figure 3: Fluorescence standard curve plotted at emission wavelength of 520 nm using different concentrations of standards.

Detailed ways

Example 1

1. GO-SELEX process for screening nucleic acid aptamers of sulfamethazine

1) Library re-denaturation treatment: Take 500nM library (5'-FAM-GACAGGCA GGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3'; N is a random sequence), put it in a metal bath and heat it at 95°C for 10 minutes, then quickly put it on ice for 10 minutes, take out After equilibrating at room temperature for 25 min in the dark, a large amount of ssDNA in the library can be folded to form a complex and diverse three-dimensional structure. and measure the fluorescence value.

2) The first round of screening: take 7μL of sulfamethazine solution (4μg/mL), add it to 200uL Lib, and incubate at 25°C, 250rpm in the dark for 1h, ssDNA self-adaptively forms a three-dimensional structure, and a part of ssDNA forms The three-dimensional structure can be combined with sulfamethazine to form the ssDNA-sulfamethazine complex. Add a certain proportion of GO and incubate at 25°C, 250rpm in the dark for 20min, free ssDNA is uniformly adsorbed on graphene oxide through π-π stacking, and the ssDNA-sulfamethazine complex cannot be bound to graphene oxide. superior. After centrifugation at 15000 rpm for 10 min at 25°C, the precipitate was discarded and the supernatant was recovered. The supernatant contains ssDNA that can bind to sulfamethazine, and the fluorescence intensity (excitation at 492 nm, emission at 520 nm) is measured on a microplate reader, and the recovery rate is calculated.

Take an appropriate amount of the supernatant collected after incubation as a template, and carry out PCR amplification with a fluorescently labeled forward primer and a biotinylated backward primer according to the above reaction conditions, and verify by polyacrylamide gel electrophoresis.

3) Calculation of recovery rate: After each round of screening and incubation, the recovery rate was calculated, recovery rate=(F _{recovered ssDNA} /F _{input ssDNA} )×100%. As the screening progresses, the ssDNA that cannot be combined with sulfamethazine is gradually screened out, and the ssDNA that can bind to sulfamethazine with high affinity is continuously enriched, and the recovery rate continues to increase until the recovery rate becomes stable, and sulfamethazine The affinity aptamers of sulfidine are enriched, and the screening process is basically over.

4) Negative screening: When the recovery rate becomes stable, a round of negative screening is carried out. After the secondary library prepared in the previous round was denatured, sulfaquinoxaline, sulfadimethoxine, and sulfamethoxine were added in an equimolar amount to the library, and incubated at 25°C, 250rpm in the dark for 1h, Graphene oxide with a mass ratio of library and graphene oxide of 1:24 was added, incubated at 25°C, 250rpm in the dark for 20min, and the ssDNA bound to sulfaquinoxaline, sulfadimethoxine, and sulfamethoxine could dissociate in the In the liquid, the ssDNA that could not be combined with them was adsorbed on graphene oxide, centrifuged at 15000 rpm, 25 °C for 10 min, and the supernatant was discarded. Add the binding buffer to the centrifuge tube containing the graphene oxide precipitate, discard the supernatant by centrifugation, repeat this operation 3 times, and completely remove the binding buffer to sulfaquinoxaline, sulfadimethoxine, and sulfamethoxine. ssDNA. Add 200 μL of binding buffer, then add equimolar amount of sulfamethazine, incubate at 25°C, 250 rpm in the dark for 1 h, the complex formed with ssDNA specifically bound to sulfamethazine is desorbed from graphene oxide, and centrifuged. Take the supernatant and measure the fluorescence intensity with a microplate reader.

5) Final round of screening: the ssDNA obtained after negative screening is repeated the positive screening process, the recovery rate is calculated, and then a round of positive screening is performed to enrich the sulfamethazine aptamer.

2. Preparation of Secondary Libraries

1) Washing magnetic beads: Take 2 tubes of 600 μL Promega magnetic beads, 1×PBS (0.1 mg/mL BSA), 1 mL/time, and wash 3 times;

2) Resuspend the magnetic beads: resuspend with 800 μL of 1×PBS (0.1 mg/mL BSA, 0.2 M NaCl, pH 7.4) respectively;

3) Extract dsDNA: add the PCR products to 800 μL of magnetic bead suspension, 25°C, 280 rpm, shake for 1 h in the dark, and the DNA is linked to the streptavidin magnet by the action of biotin and streptavidin. on beads

4) Separation of dsDNA: use a magnetic stand to separate and remove the supernatant, and wash three times with 1 mL of 1×PBS (pH 7.4) to remove DNA that is not bound to the magnetic beads, and finally combine two tubes of magnetic beads to remove the supernatant;

5) ssDNA regeneration: add 50 μL of 0.05M NaOH, and vortex for 2 min to separate the sense and antisense strands under alkaline conditions. The ssDNA strand with biotin is attached to the magnetic beads, and the other without biotin After the chain alkali was cleaved, 25 μL of ddH ₂ O and 100 μL of 2×Tris-HCl were added and vortexed for 1 min. The supernatant was carefully collected by magnetic separation, and 25 μL of 0.1M HCl was added to the collected solution to neutralize NaOH to obtain 200 μL of secondary library. Finally, the fluorescence intensity (excitation at 492 nm, emission at 520 nm) was measured with a microplate reader, and the measurement was repeated three times, and the concentration of the secondary library was calculated according to the standard curve. The secondary library was appropriately diluted and processed according to the first round of repeated denaturation, and sulfamethazine was added to enter the next round of screening.

3. Cloning and Sequencing

The ssDNA obtained after 7 rounds of screening was amplified by PCR using unlabeled forward primers 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

First, use the software DNAMAN to perform sequence alignment and homology analysis on the sequences obtained by sequencing, and analyze the characteristics of each nucleic acid sequence; then use Mfold to predict the secondary structure of the nucleic acid sequence online; determine the affinity of No 1 and No 5, and then according to The nucleic acid sequence was appropriately truncated by the secondary structure, and the affinity was improved, and two trimmed nucleic acid aptamers No 1s and No 5s were obtained, as shown in Table 2 for details.

Table 2

No1s CGTTAGACG No5s GCTGATAGC

5. Affinity Determination

Graphene oxide can not only adsorb ssDNA, but also has a good fluorescence quenching effect, which can quench fluorescent groups. Gradient concentrations (12.5, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0 nmol/L) of 5'-FAM fluorescently labeled candidate aptamers were mixed with equimolar amounts of sulfamethazine, respectively, and incubated at 25°C in the dark 1h. Then, graphene oxide solutions were added with different ssDNA/GO mass ratios, mixed well, and incubated at 25 °C for 20 min in the dark. Centrifuge at 15000rpm, 25°C for 10min, draw the supernatant containing the sulfamethazine-aptamer complex, and measure its fluorescence intensity with a microplate reader (excitation at 492 nm, emission at 520 nm). At the same time, each concentration of candidate aptamer sequences was incubated with different mass ratios of GO (without target sulfamethazine) as a negative control to eliminate the effect of self-desorption of candidate aptamer sequences from the surface of GO. GraphPad Prism 5.0 software was used to perform nonlinear regression analysis on each sequence, and the concentration of candidate aptamers was used as the abscissa, and the difference in fluorescence intensity (ΔF) between the experimental group and the control group was used as the ordinate to draw the binding saturation curve, and calculate the dissociation. Constant (K _d ). As shown in Figure 1, the affinities of No 1, No 5, No 1s, and NO 5s are: 167.8nM, 185.7nM, 76.4nM, and 81.3nM, respectively.

Nucleic acid aptamer derivatives obtained by modifying and transforming the above DNA fragments also belong to the protection scope of the present invention.

6. Binding specificity experiments of nucleic acid aptamers and sulfamethazine

Take sulfa drugs similar to sulfamethazine: sulfadimethoxine, sulfamethoxine, sulfaquinoxaline, and different antibiotics nitrofuran, samizuril, and the specificity of the screened aptamers to measure. 100 nmol/L aptamer was combined with sulfamethoxine, sulfaquinoxaline, sulfadimethoxine, nitrofuran and samizuril respectively for 60 min, added graphene oxide in the same mass ratio, and incubated with slight shaking for 20 min , the fluorescence intensity of the supernatant was measured with a microplate reader, and the specificity of the nucleic acid aptamer was analyzed by the fluorescence intensity of the candidate drug (emission wavelength 520 nm). As shown in Figure 2, compared with other antibiotics, the fluorescence intensity of sulfamethazine is the highest, indicating that the No 1s aptamer has high specificity for sulfamethazine.

7. Standard curve establishment

FAM fluorescently labeled aptamers (100 nM) were incubated with a range of concentrations ranging from 0.4-500 ng/mL sulfamethazine in 200 μL of binding buffer for 1 h at 25 °C in the dark, GO was added to the mixture and incubated at room temperature in the dark After 20min, the fluorescence intensity was measured by the microplate reader at the emission wavelength of 520nm, and a standard curve was drawn. Figure 3 is the relationship between the fluorescence intensity of No 1s aptamer at different concentrations of sulfamethazine (0.4ng/mL～500ng/mL).

Embodiment 2 Determination of actual sample sulfamethazine

Milk and egg samples purchased from a local supermarket, previously analyzed by HPLC, were confirmed to be free of SMZ compounds. A 10 ml milk sample was taken and first centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was diluted to 100 mL with binding buffer and filtered through a 0.22-μm microporous filter. For egg samples, after thorough mixing and homogenization, add 2 g of egg sample to the centrifuge tube. Then 4 mL of ethyl acetate was added and shaken for 3 min. After centrifugation at 5000 × g for 5 min at room temperature, the supernatant was dried in a water bath at 80°C or in nitrogen, and diluted to 500 μL with binding buffer. To evaluate the recovery of the constructed graphene oxide-based fluorescent aptamer sensor, samples were spiked with different concentrations of sulfamethazine (2.0, 10.0, 25.0, 50.0, and 100.0 ng/mL), respectively, and the constructed aptamer sensor to detect. Draw the curve according to the operation method of step 7, and carry out the experiment and analysis of the results.

The lower limit of detection (LOD) is calculated as: LOD=3*SD/slope, where SD represents the standard deviation of the fluorescence values of 20 blank samples (milk and eggs) respectively. Substitute into the equation to calculate the sample detection limit. Experimental results: In the test of sulfamethazine in milk and egg samples, the detection limits of the samples were 1.37 μg/kg and 3.15 μg/kg, respectively.

The recovery rates are shown in Table 3. The recovery rates of milk samples are between 94.4% and 108.8%, and the recovery rates of egg samples are between 93.9% and 106.7%. The detection coefficients of variation in milk and eggs by this method ranged from 5.4 to 12.7 and 5.1 to 8.8, respectively. According to the results of coefficient of variation and recovery rate, the method has good repeatability and accuracy, and can meet the needs of on-site detection of food and medicine. See Table 3 for details.

Table 3. Accuracy and precision of addition of sulfamethazine to eggs and milk (n=5).

sequence listing

<110> Chongqing Normal University

<120> A sulfamethazine nucleic acid aptamer screening method, kit and application

<130> 2019

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 80

<212> DNA

<213> No.1

<400> 1

gacaggcagg acaccgtaac gttagacgct ggaccgcgtg tgatcgggtc agctgcaatt 60

ctgctacctc cctcctcttc 80

<210> 2

<211> 80

<212> DNA

<213> No.2

<400> 2

gacaggcagg acaccgtaac gctctgggct ggcgatgggc agtgttaaat ttgtgccacc 60

ctgctacctc cctcctcttc 80

<210> 3

<211> 80

<212> DNA

<213> No.3

<400> 3

gacaggcagg acaccgtaac gcttgggctg gggatgcccg ataaggagat gcgtccacgc 60

ctgctacctc cctcctcttc 80

<210> 4

<211> 80

<212> DNA

<213> No.4

<400> 4

gacaggcagg acaccgtaac tgatgtcgat cgtcacttcg acgggataaa cggtataaga 60

ctgctacctc cctcctcttc 80

<210> 5

<211> 80

<212> DNA

<213> No.5

<400> 5

gacaggcagg acaccgtaac taccgggtaa tatgcacaca tgctgatagc aagcaatgca 60

ctgctacctc cctcctcttc 80

<210> 6

<211> 80

<212> DNA

<213> No.6

<400> 6

gacaggcagg acaccgtaac ttacctctct gaggtttcgg ttaggggaag tatagactga 60

ctgctacctc cctcctcttc 80

<210> 7

<211> 9

<212> DNA

<213> No.1s

<400> 7

cgttagacg 9

<210> 8

<211> 9

<212> DNA

<213> No.5s

<400> 8

gctgatagc 9

Claims (8)

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

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

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.

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.

Images