CN114058624A - Aptamer, sensor, kit and application for detecting sulfamoyl-5-methoxypyrimidine - Google Patents

Abstract

The invention discloses an aptamer, a sensor, a kit and application for detecting sulfanilamide-5-methoxypyrimidine. The sequence of the aptamer is SME4-1 (sequence number: CCGACTGGCTCGG) recorded in a sequence table, and an aptamer derivative obtained by modifying or modifying the aptamer. The invention obtains an aptamer of sulfanilamide-5-methoxypyrimidine by a fixed magnetic bead-SELEX screening method; selecting a sequence with high affinity by predicting the obtained aptamer secondary structure; by means of affinity determination, the aptamer with the highest affinity is subjected to sequence truncation to obtain the core recognition area of the aptamer and obtain the optimal aptamer. The invention also provides aptamers and Fe₃O₄/Au/g‑C₃N₄The fluorescent aptamer sensor is obtained by mixed construction, and the sulfanilamide-5-methoxypyrimidine detection kit containing the sensor. The fluorescence detection kit is used for detecting the sulfamoyl-5-methoxypyrimidine, and has the advantages of high sensitivity, simple operation, strong specificity, high speed and low price. Has good application prospect in the detection of the sulfamoyl-5-methoxy pyrimidine.

Description

Aptamer, sensor, kit and application for detecting sulfamoyl-5-methoxypyrimidine

Technical Field

The invention relates to the field of antibiotic residue detection in biotechnology, in particular to an aptamer, a sensor, a kit and application for detecting sulfanilamide-5-methoxypyrimidine.

Background

Antibiotics are widely used in animal husbandry and in drugs for treating diseases in livestock and for promoting the growth of farmed animals. Antibiotics are used in excess of millions of tons in the world, and sulfonamides are used in excess of 20% of the total amount, so that the antibiotics can be used as cheap and effective medicaments for resisting gram-negative bacteria and gram-positive bacteria. Sulfamethoxazole (sulfamethoxazine) belongs to the Sulfonamide (SAs) family and is one of the most commonly used antibiotics in livestock due to its broad spectrum of activity and low cost. However, abuse by small and medium-sized enterprises leads to overproof residues in animal-derived foods and their transmission to the human body through the food chain. Excessive intake of sulfamoyl-5-methoxypyrimidine in humans can cause allergic and toxic reactions. The Maximum Residual Limit (MRL) of total sulpha-5-methoximine in foods of animal origin has been defined in the European Union, the United states and China not to exceed 100 mug/kg. Therefore, it is urgent to develop a method for detecting sulfanilamide-5-methoxylpyrimidine with high sensitivity and high specificity that satisfies the requirements of food detection.

The detection methods of the sulfanilamide-5-methoxypyrimidine are more, and comprise high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, capillary zone electrophoresis, chemiluminescence enzyme immunoassay and enzyme-linked immunosorbent assay. The high performance liquid chromatography is the most common method for detecting sulfonamides at present, and has higher sensitivity and specificity. However, high performance liquid chromatography and liquid chromatography-tandem mass spectrometry require expensive experimental equipment, require specialized operators to perform, and are not conducive to the detection of large numbers of samples. Capillary zone electrophoresis also requires expensive laboratory equipment and is cumbersome to operate. Chemiluminescence enzyme immunoassay and enzyme-linked immunosorbent assay are limited by the preparation of antibody, and the performance and specificity of the antibody determine the accuracy of the determination method. These conventional methods do not meet the detection requirements of wider application, simpler and more economical.

In recent years, a novel nucleic acid probe has emerged in the field of detection: nucleic acid aptamers (aptamers for short). Aptamers, also known as "chemical antibodies", have a number of advantages over, for example, traditional antibodies, such as: stable chemical property, easy modification, short screening time, no need of animal experiment, in vitro screening, etc. Aptamers have been obtained by systematic evolution of exponential enrichment ligands (SELEX), have oligonucleotide sequences of 70nt to 100nt, have high affinity and high specificity, are capable of specifically binding to target molecules, and have been used for the detection of small molecule substances, such as: metal ions, antibiotics, toxins and macromolecular substances, such as: cells, viruses, proteins, etc. The aptamer is widely applied to the fields of biological medicine industry, food environment detection and the like. It has been demonstrated that aptamers can fold into unique spatial structures under specific buffer systems, which can bind specifically and tightly to target molecules through van der waals forces, hydrogen bonding, and hydrophobic interactions.

At present, no test report of the application of the sulfanilamide-5-methoxypyrimidine aptamer to sulfanilamide-5-methoxypyrimidine exists.

Disclosure of Invention

In view of the above, in order to overcome the defects of the prior art, the invention provides an FAM fluorescence labeled sulfanilamide-5-uracil aptamer, which has high affinity and specificity to sulfanilamide-5-uracil and is suitable for qualitative or quantitative detection of sulfanilamide-5-uracil in various environments and mediums.

The invention provides an FAM fluorescent labeling aptamer for detecting sulfanilamide-5-methoxypyrimidine, wherein the sequence of the aptamer is as shown in SEQ No. 1: SME4, SEQ No. 2: SME9, SEQ No. 3: SME15, SEQ No. 4: SME26, SEQ No. 5: SME50, SEQ No. 6: SME54 or SEQ No. 7: SME4-1, and aptamer derivatives obtained by modifying or modifying the aptamer.

Further, the FAM fluorescent labeling aptamer sequence for detecting the sulfanilamide-5-methoxypyrimidine is SME4-1 recorded in a nucleotide sequence table.

The nucleotide aptamer consists of single-stranded DNA, and the 5' end is labeled with FAM fluorescent group.

The invention relates to a preparation method of FAM fluorescent labeling aptamer for detecting sulfanilamide-5-methoxypyrimidine, which comprises the following steps:

1) obtaining an aptamer of the sulfanilamide-5-methoxypyrimidine by a fixed magnetic bead-SELEX screening method;

2) selecting a sequence with high affinity by predicting the obtained aptamer secondary structure;

3) by means of affinity determination, the aptamer with the highest affinity is subjected to sequence truncation to obtain the core recognition area of the aptamer and obtain the optimal aptamer.

The invention also provides Fe-based method for detecting the sulfamoyl-5-methoxypyrimidine₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor of (a), the sensor composition comprising:

(1) FAM-SME4-1 described in the FAM fluorescent labeled nucleotide sequence table;

(2)Fe₃O₄/Au/g-C₃N₄；

(3) binding buffer solution (100mM NaCl,2mM MgCl)₂,20mM Tris-HCl,1mM CaCl₂,5mM KCl, and 0.02％Tween 20,pH 7.6)。

The invention also provides a kit for detecting the sulfamoyl-5-methoxypyrimidine, which comprises the Fe-based kit₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor of (1).

The invention provides a kit for detecting sulfanilamide-5-methoxypyrimidine, which comprises the following components:

(1) FAM-SME4-1 described in FAM fluorescence labeling nucleotide sequence table;

(2)Fe₃O₄/Au/g-C₃N₄；

(3) binding buffer solution (100mM NaCl,2mM MgCl2,20mM Tris-HCl,1mM CaCl2,5mM KCl, and 0.02% Tween 20, pH 7.6);

(4) and a magnet.

The use method of the kit comprises the following steps:

1) treating each animal-derived food sample according to the national standard requirements;

2) mixing the treated sample with FAM-SME4-1, and shaking for 30 minutes;

3) adding Fe₃O₄/Au/g-C₃N₄Continuously shaking for 5 minutes;

4) after magnetic separation the supernatant was aspirated and fluorescence was measured.

The invention also provides application of the kit for detecting the sulfanilamide-5-methoxypyrimidine in quantitative detection of the sulfanilamide-5-methoxypyrimidine in animal-derived tissue test samples.

The invention provides a sulfanilamide-5-methoxypyrimidine fluorescence detection kit, which comprises a Fe-based fluorescence detection kit₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor of (a); wherein the fluorescent aptamer sensor comprises Fe₃O₄/Au/g-C₃N₄And FAM-labeled aptamers; the sequence of the FAM-labeled aptamer is one of sulfanilamide-5-methoxy pyrimidine 4, sulfanilamide-5-methoxy pyrimidine 9, sulfanilamide-5-methoxy pyrimidine 15, sulfanilamide-5-methoxy pyrimidine 26, sulfanilamide-5-methoxy pyrimidine 50, sulfanilamide-5-methoxy pyrimidine 54 or sulfanilamide-5-methoxy pyrimidine 4-1 which are described in a nucleotide sequence table. The modified and modified aptamer derivative also belongs to the protection scope of the invention.

The application of the sulfanilamide-5-methoxypyrimidine fluorescence detection kit comprises the following steps: the application of the quantitative detection of the sulfanilamide-5-methoxypyrimidine in samples such as milk, honey, eggs, pork, beef, chicken, pork liver, chicken liver, crucian, shrimp and the like.

Compared with the prior art, the invention has the beneficial effects that:

1. the biosensor constructed based on the aptamer is used for detecting the sulfamoyl-5-methoxypyrimidine, and has the advantages of high sensitivity, simplicity in operation, strong selectivity, high speed and low price. Has good application prospect in the detection of the sulfamoyl-5-methoxy pyrimidine.

2. The invention obtains the core recognition area of the aptamer by analyzing and optimizing the aptamer sequence obtained by screening the magnetic bead-SELEX, and synthesizes the aptamer with high affinity and specificity to the sulfanilamide-5-methoxypyrimidine by in vitro cloning.

3. The invention relates to an aptamer and Fe based on sulfanilamide-5-methoxy pyrimidine₃O₄/Au/g-C₃N₄A fluorescent aptamer sensor was constructed. When the sulfanilamide-5-methoxy pyrimidine exists in the detection system, the aptamer is specifically combined with the sulfanilamide-5-methoxy pyrimidine and cannot be accumulated by Fe through pi-pi₃O₄/Au/g-C₃N₄And (4) adsorbing. And the fluorescence values detected by the concentration of the sulfanilamide-5-methoxylpyrimidine are different. When the detection system does not contain the sulfanilamide-5-methoxy pyrimidine, the fluorescence labeled aptamer is accumulated by pi-pi and is Fe₃O₄/Au/g-C₃N₄And (4) adsorbing. Based on the principle, the quantitative detection of the sulfanilamide-5-methoxypyrimidine is realized.

4. The invention aims at the problems that the traditional detection method can not detect large-scale samples and has complex operation and long-time animal experiments are needed for preparing the antibody in the immunoassay method, and establishes the Fe-based method₃O₄/Au/g-C₃N₄The rapid detection method of the fluorescence sensor can be used for rapid and sensitive quantitative detection of the residue of the sulfamethoxazole-5-methoxylpyrimidine in the animal derived food, and overcomes the defects of the method. Provides a new detection method for detecting antibiotics in animal-derived food.

5. The invention is based on Fe₃O₄/Au/g-C₃N₄The sulfonamide-5-methoxyl pyrimidine aptamer is screened and optimized in sequence, and the Fe-based aptamer is constructed₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor successfully detects in actual samples of milk and eggs, has good linear relation, and lays a foundation for detecting and developing products of the sulfanilamide-5-methoxypyrimidine.

Drawings

FIG. 1: the invention is based on Fe₃O₄/Au/g-C₃N₄Schematic diagram of the fluorescent aptamer sensor of (1);

FIG. 2: determination of aptamer affinity;

FIG. 3: specific analysis of fluorescent aptamer sensors;

FIG. 4: and (5) drawing a standard curve.

Detailed Description

Example 1 establishment of a sulfonamide-5-methoxypyrimidine aptamer assay System

1. Magnetic bead-SELEX process for screening sulfonamide-5-methoxypyrimidine aptamers

1) Library and primer treatment: 200 μ L of 500nM aptamer library (5 '-FAM-CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTG C-3', N is random sequence), 100 μ L of 10 μ M forward primer (5'-CACCTAATACGACTCACTATAGCGGA-3') and 100 μ L of 1 μ M reverse primer (5'-GCAAGCTTGTTCGAGCCAG-3') and 100 μ L of 10 μ M Biotin-reverse primer (Biotin-5'-GCAAGCTTGTTCGAGCCAG-3') were placed in a metal bath at 95 ℃ for 10 minutes, followed by ice for 30 minutes, after which they were kept in the dark at room temperature to allow a large amount of ssDNA in the library to fold to form unique tertiary structures, and fluorescence was measured.

2) Preparing coupling magnetic beads and purified magnetic beads:

coupling magnetic beads: mu.L of 10mg/mL magnetic carboxyl beads were washed four times with DMF and collected using a magnet. The magnetic beads were reacted with 100. mu.L of 5. mu.M HATU and 100. mu.L of 3. mu.M DIPEA for 2 hours at 25 ℃ and then 200. mu.L of 1mg/mL sulfanilamide-5-methoxypyrimidine was added to the mixture and shaken overnight at 25 ℃. Subsequently, binding buffer (100mM NaCl,2mM MgCl) was used₂、20mM Tris-HCl、1mM CaCl₂5mM KCl and 0.02% Tween 20, pH 7.6) the magnetic beads were washed 4 times. The resulting beads were stored at 4 ℃ until use. In addition, sulfadimidine, sulfaquinoxaline and sulfadiazine were also separately coupled to the magnetic beads using the same procedure as described above.

Purifying magnetic beads: to obtain purified magnetic beads, 1mL of 1mg/mL streptavidin magnetic beads in PBS (pH 7.4) was reacted with 100. mu.L of 10. mu.M biotin-reverse primer at 25 ℃ for 20 minutes and washed 4 times with PBS (pH 7.4). The collected magnetic beads were stored in PBS (pH 7.4) at 4 ℃ until use.

3) Obtaining the sulfanilamide-5-methoxyl pyrimidine aptamer through magnetic bead-SELEX screening: 100 μ L of the carboxyl magnetic beads were taken, the supernatant removed and 100 μ L of 500nM aptamer library added, followed by incubation at 25 ℃ for 1 hour. The magnetically separated supernatant was collected and used as a new library for forward selection. Then 100. mu.L of magnetic beads coupled with sulfanilamide-5-methoxypyrimidine was taken, and after removing the supernatant, 100. mu.L of a new library was added and incubated at 25 ℃ for 1 hour. The sulfonamide-5-methoxypyrimidine-coupled magnetic beads were washed 4 times with the binding buffer, and the resulting supernatant was removed completely. Subsequently, the sulfonamide-5-methoxypyrimidine coupled magnetic beads were washed with 200. mu.L of an elution buffer (40mM Tris-HCl, 3.5M urea, 10mM EDTA, 0.02% Tween 20, pH 8.0), incubated at 80 ℃ for 10 minutes, and then the eluate was collected by magnetic separation. This elution procedure was repeated 4 times.

The collected 800. mu.L eluate and purified magnetic beads were incubated at 25 ℃ for 1 hour, then washed 2 times with PBS (pH 7.4) and the supernatant removed, then eluted with 50. mu. L0.05M NaOH, and after incubation at 25 ℃ for 5 minutes the eluate (. about.50. mu.L) was collected by magnetic separation, and the pH was adjusted to 7.4 using 0.05M HCl. Then 2 Xbinding buffer (175 mM NaCl, 4mM MgCl)₂、40mM Tris-HCl、2mM CaCl₂10mM KCl and 0.04% Tween 20, pH 7.6) equilibrating the buffer system in which the aptamer resides.

4) The resulting ssDNA library in the eluate was used for PCR amplification:

PCR procedure: mu.L of ssDNA library, 2. mu.L of 10. mu.M forward primer, 2. mu.L of 1. mu.M reverse primer, 5. mu.L of 2 × taq PCR MasterMix II and 36. mu.L of ddH₂O-mix, denaturation at 95 ℃ for 30s, renaturation at 58 ℃ for 30s and extension at 72 ℃ for 6 s. This amplification process was repeated for 20 cycles. The amplification product was purified using the same procedure as in step 3) above and used as an aptamer library for the next round.

5) And carrying out reverse screening by using sulfamethazine, sulfaquinoxaline and sulfadiazine-magnetic beads in the seventh round, and then continuing forward screening by using sulfa-5-methoxypyrimidine-magnetic beads. UV-VIS spectroscopy was used to determine the aptamer concentration at the end of each round of screening.

6) Determination of recovery: (each round of purification recovery ssDNA concentration/start of the round of ssDNA concentration) 100%.

2. Cloning and sequencing

After ten rounds of screening, the obtained product is amplified by PCR, and a clone is selected for sequencing.

3. Aptamer sequence analysis and optimization

Predicting the secondary structure of the obtained aptamer through DANMAN, RNAscope, Vienna RNA Web Services and UNAFold Web Server; the sequences that meet the requirements were selected and are shown in Table 1. The affinity of the candidate aptamer is then determined by an affinity assay. Truncating the sequence of the aptamer with the highest affinity to obtain a new aptamer: SME4-1, the sequence is shown in Table 2.

TABLE 1

TABLE 2

4. Affinity assay

To assess the affinity of candidate aptamers, 100 μ L of SME-carboxy functionalized magnetic beads (10mg/mL) were mixed with 200 μ L of different concentrations of FAM-labeled aptamers (i.e., 0, 25, 50, 100, 200, and 400nM), the mixture was allowed to react for 1 hour at 25 ℃ in the dark, and then the reaction mixture was washed twice with binding buffer. Subsequently, 100. mu.L of 50mM NaOH was added to the resulting magnetic beads, and incubated for 5 minutes with gentle stirring. Finally, the supernatant from the reaction mixture was collected and used with a fluorimeter at λ_ex492nm and λ_emThe fluorescence intensity was measured at 518 nm. K representing binding affinity_dValues were analyzed using Origin 2019 software and according to the nonlinear regression equation Y ═ B_max×X÷(K_d+ X) calculation, where X represents aptamer concentration and Y represents relative fluorescenceLight intensity, B_maxRepresenting the most binding sites. As shown in table 3 (fig. 2), the affinities of SME4, SME9, SME19, SME26, SME50, SME56 and SME4-1 were 315.77, 401.07, 357.22, 505.93, 446.78, 405.26 and 83.65nM, respectively.

TABLE 3

Aptamer derivatives obtained by modifying and modifying the above aptamers also belong to the scope of the present invention.

5.Fe₃O₄/Au/g-C₃N₄Preparation of

15 g of urea were heated at 550 ℃ for 4 hours, and at the end of the reaction, yellow g-C was obtained₃N₄. 0.5g g-C₃N₄Added to 25mL trisodium citrate solution (1.5mM) and sonicated for 20 minutes. To the reaction mixture was added 25mL of a chloroauric acid solution (1mM), and the resulting mixture was reacted at 60 ℃ for 2 hours under constant stirring. Subsequently, the reaction mixture was washed 4 times with deionized water, absolute ethanol, respectively, and then dried at 60 ℃ to obtain Au/g-C₃N₄. 0.15 g of Au/g-C₃N₄、0.135g FeCl₃·6H₂O and 0.2025g FeSO₄·7H₂O dissolved in 50mL DI H₂In O, ultrasonic treatment is carried out for 1 hour. Subsequently, 0.12g of NaOH was dissolved in the solution, and the resulting solution was transferred to a hydrothermal reactor and the reaction was carried out at 120 ℃ for 24 hours. Washing the reaction product with deionized water and absolute ethyl alcohol respectively for 4 times to obtain brownish black magnetic material Fe₃O₄/Au/g-C₃N₄。

6. Specificity analysis of aptamer and sulfanilamide-5-methoxypyrimidine

To evaluate the specificity of aptamers to sulfanilamide-5-methoxypyrimidine, 200. mu.L of a reaction mixture was prepared in which the concentration of fluorescently labeled aptamers was 100nM and sulfanilamide-5-methoxypyrimidine or its structural analogs (sulfaquinoxaline, sulfadiazine, sulfadimidine, sulfadimethoxine, sulfadimidine)Methyloxazole, sulfapyridine, nitrofurantoin, pyrimethamine and kanamycin) to a final concentration of 1. mu.g/. mu.L. 30 μ L of 2mg/mL Fe₃O₄/Au/g-C₃N₄Adding into the mixture, mixing, and adsorbing Fe with magnet₃O₄/Au/g-C₃N₄I.e., after magnetic separation, the supernatant was aspirated and fluorescence was measured (as shown in fig. 1). The specificity of the aptamers was evaluated by the relative ratio of fluorescence intensity (measured fluorescence intensity of other antibiotics/measured fluorescence intensity of sulfanilamide-5-methoxypyrimidine) x 100%. As shown in FIG. 3, the relative fluorescence intensity of other antibiotics and the structural analogue of sulfamonomethoxine is less than 25%. These results indicate that SME4-1 has a high specificity for sulfamoyl-5-methoxypyrimidine.

7. Establishment of a Standard Curve

A standard curve was constructed using a sulfonamide-5-methoxypyrimidine standard, and FAM fluorescently labeled aptamer (100nM) was incubated with a series of 2-250ng/mL sulfam-5-methoxypyrimidine in 200. mu.L of binding buffer in the dark at 25 ℃ for 1h, Fe₃O₄/Au/g-C₃N₄The mixture was added, incubated for 5 minutes at room temperature in the dark, and the fluorescence intensity was measured at an emission wavelength of 520nm with a microplate reader, and a standard curve was plotted (shown in FIG. 4).

EXAMPLE 2 determination of the actual sample sulfanilamide-5-methoxypyrimidine

The aptamer sensors were validated using 10 different samples purchased from the local market, after which HPLC was used to confirm that these samples did not contain sulfamoyl-5-methoxypyrimidine. 10mL skim milk was centrifuged at 14000rpm for 20 minutes at 4 ℃ and the supernatant was collected and diluted to 100 mL. Finally, the mixture was filtered through a 0.22 μm filter. 2g of eggs and 4ml of ethyl acetate were shaken for 10 minutes, then centrifuged at 5000 rpm for 5 minutes, and finally the ethyl acetate was removed at 40 ℃ using a metal bath nitrogen blower. Honey was diluted 10-fold with PBS to eliminate matrix interference. Pig, pork liver, chicken liver, cattle, crucian and shrimp are mashed by a meat grinder and homogenized by dispersing emulsifier by a homogenizer (for crucian, peeling, for shrimp, shelling and for shrimp, only taking shrimp meat from the digestive tract of the shrimp). 5g of the treated sample was added to 25mL of acetonitrile and shaken for 15 minutes. The mixture was sonicated for 10 minutes and centrifuged at 5000 Xg for 15 minutes. The supernatant was collected and 30mL of acetonitrile saturated n-hexane was added and mixed for 10min to remove fats. The liquid in the acetonitrile layer was collected and then heated in a water bath at 80 ℃ to remove acetonitrile. The resulting product was dissolved in binding buffer and diluted 10-fold for use.

The accuracy and precision of the aptamer sensor are expressed in terms of recovery and coefficient of variation, respectively. Known concentrations of sulfamoyl-5-methoxypyrimidine (50, 100 and 150. mu.g/kg) were added to ten samples and subjected to aptamer sensor and HPLC analysis, respectively. The average recovery is calculated by the following equation: (measured concentration/spiked concentration). times.100%. The coefficient of variation was determined by analyzing the above samples with three different levels of sulfamoyl-5-methoxypyrimidine added. The calculation formula of the coefficient of variation: coefficient of variation CV ═ standard deviation/average × 100%. Five tests were performed per concentration level and the correlation of aptamer sensor and HPLC analysis in the detection of sulfamethoxine in spiked samples was calculated.

As shown in Table 4, the recovery of the sample was between 92.42% and 107.22% with a coefficient of variation between 1.43% and 14.46%. A positive correlation between the results of the aptamer sensor and HPLC was also observed (R)²>0.9304). The results show the reliability of the proposed aptamer sensor for detecting sulfamoyl-5-methoxypyrimidine.

Table 4: the mean recovery and coefficient of variation for the spiked samples and the correlation between aptamer sensor and HPLC results (n-5).

Embodiment 3 the kit for detecting sulfamoyl-5-methoxypyrimidine provided by the present invention:

the kit comprises the following components:

(1) FAM-SME4-1 described in a 100nM FAM fluorescence labeling nucleotide sequence table;

(2)2mg/mLFe₃O₄/Au/g-C₃N₄；

(3) combined with a buffer solutionLiquid (100mM NaCl,2mM MgCl2,20mM Tris-HCl,1mM CaCl)₂,5mM KCl, and 0.02％Tween 20,pH 7.6)；

(4) And a magnet.

The detection is carried out according to the following steps:

1) treating each animal-derived food sample as required;

2) the treated sample was dissolved in binding buffer and diluted 10-fold. Adding 100nM FAM-SME4-1, mixing and shaking for 30 min;

3) 30 μ L of 2mg/mL Fe was added₃O₄/Au/g-C₃N₄Continuously shaking for 5 minutes;

4) magnet adsorbing Fe₃O₄/Au/g-C₃N₄I.e., after magnetic separation, the supernatant was aspirated and fluorescence was measured.

The specific examination procedure was the same as in example 2.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

SEQUENCE LISTING

<110> university of Chongqing teacher

Shenzhen Institute of Information Technology

<120> aptamer, sensor, kit and application for detecting sulfanilamide-5-methoxypyrimidine

<130> CN 107119054 A

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Claims (9)

1. A FAM fluorescence labeling aptamer for detecting sulfanilamide-5-methoxypyrimidine is characterized in that: the sequence of the aptamer is SEQ No. 1: SME4, SEQ No. 2: SME9, SEQ No. 3: SME15, SEQ No. 4: SME26, SEQ No. 5: SME50, SEQ No. 6: SME54 or SEQ No. 7: SME4-1, and aptamer derivatives obtained by modifying or modifying the aptamer.

2. The FAM fluorescently labeled aptamer for detecting sulfam-5-methoxypyrimidine according to claim 1, characterized in that: the nucleotide aptamer consists of single-stranded DNA, and the 5' end is labeled with FAM fluorescent group.

3. The method for preparing FAM fluorescent labeled aptamer for detecting sulfamoyl-5-methoxypyrimidine according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

1) obtaining an aptamer of the sulfanilamide-5-methoxypyrimidine by a fixed magnetic bead-SELEX screening method;

2) selecting a sequence with high affinity by predicting the obtained aptamer secondary structure;

3) by means of affinity determination, the aptamer with the highest affinity is subjected to sequence truncation to obtain the core recognition area of the aptamer and obtain the optimal aptamer.

4. Based on Fe₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor of (a), wherein the sensor composition comprises:

(1) FAM-SME4-1 described in FAM fluorescence labeling nucleotide sequence table;

(2)Fe₃O₄/Au/g-C₃N₄；

(3) the buffer solution is combined.

5. A kit for detecting sulfanilamide-5-methoxylpyrimidine is characterized in that: comprising the Fe-based alloy of claim 4₃O₄/Au/g-C₃N₄The fluorescent aptamer sensor of (1).

6. The kit for detecting sulfam-5-methoxypyrimidine according to claim 5, characterized in that: the kit comprises the following components:

(1) FAM-SME4-1 described in FAM fluorescence labeling nucleotide sequence table;

(2)Fe₃O₄/Au/g-C₃N₄；

(3) combining with a buffer solution;

(4) and a magnet.

7. The method of using the kit of claim 6, wherein: the method comprises the following steps:

1) treating each animal-derived food sample as required;

2) mixing the treated sample with FAM-SME4-1, and shaking for 30 minutes;

3) adding Fe₃O₄/Au/g-C₃N₄Continuously shaking for 5 minutes;

4) after magnetic separation the supernatant was aspirated and fluorescence was measured.

8. The use of the kit for the detection of sulfamonomethoxine of claim 6 for the quantitative detection of sulfamonomethoxine in animal derived tissue test samples.

9. The use of claim 8, wherein the tissue test sample of animal origin is milk, honey, egg, pork, beef, chicken, pork liver, chicken liver, crucian or shrimp.

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