CN114058624B

CN114058624B - Aptamer, sensor, kit and application for detecting sulfanilamide-5-methoxypyrimidine

Info

Publication number: CN114058624B
Application number: CN202111484999.8A
Authority: CN
Inventors: 乐涛; 严雪凌; 时婧婧; 孙琦
Original assignee: Chongqing Normal University; Shenzhen Institute of Information Technology
Current assignee: Chongqing Normal University; Shenzhen Institute of Information Technology
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2023-06-23
Anticipated expiration: 2041-12-07
Also published as: CN114058624A

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 modification or transformation of the aptamer. According to the invention, an aptamer of the sulfa-5-methoxypyrimidine is obtained through a fixed magnetic bead-SELEX screening method; selecting a sequence with high affinity through predicting the secondary structure of the obtained aptamer; and (3) carrying out sequence truncation on the aptamer with the highest affinity through affinity measurement, so as to obtain a core recognition region of the aptamer, and obtaining the optimal aptamer. The invention also provides an aptamer and Fe ₃ O ₄ /Au/g‑C ₃ N ₄ And mixing the fluorescent aptamer sensor obtained by the construction and a sulfanilamide-5-methoxypyrimidine detection kit containing the sensor. The fluorescence detection kit is used for detecting the sulfanilamide-5-methoxypyrimidine, and has the advantages of high sensitivity, simplicity in operation, strong specificity, high speed and low cost. Has good application prospect in the detection of sulfanilamide-5-methoxypyrimidine.

Description

Aptamer, sensor, kit and application for detecting sulfanilamide-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 the livestock industry and as medicines for treating livestock diseases and promoting the growth of farmed animals. Antibiotics are used in amounts exceeding millions of tons worldwide, and sulfonamides are used in amounts exceeding 20% of the total amount, and can be used as inexpensive and effective agents against gram-negative and gram-positive bacteria. Sulfa-5-methoxypyrimidine (sulfamethoxydiazine) belongs to the family of Sulfa Antibiotics (SAs), which are among the most commonly used antibiotics in livestock due to its broad spectrum of activity and low cost. However, abuse by small and medium enterprises leads to exceeding of their residues in animal-derived foods and transmission to the human body through the food chain. Excessive intake of sulfa-5-methoxypyrimidine in humans causes allergic and toxic reactions. The European Union, the United states and China have stipulated that the Maximum Residual Limit (MRL) of total sulfonamide-5-methoxypyrimidine in animal derived foods should not exceed 100 μg/kg. Therefore, it is an urgent need to develop a highly sensitive and highly specific method for detecting sulfanilamide-5-methoxypyrimidine that meets the food detection requirements.

The detection method of the sulfanilamide-5-methoxypyrimidine is more, and comprises 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 commonly used method for detecting the 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 disadvantageous for detection of large amounts of samples. Capillary zone electrophoresis also requires expensive laboratory equipment and is cumbersome to operate. Chemiluminescent enzyme immunoassays and enzyme-linked immunosorbent assays are limited by the preparation of antibodies, the properties and specificity of which determine the accuracy of the assay. None of these commonly used methods meet the detection requirements of broader, simpler, and more economical application.

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

At present, no detection report of sulfa-5-methoxypyrimidine aptamer for sulfa-5-methoxypyrimidine exists.

Disclosure of Invention

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

The invention provides a FAM fluorescent labeling aptamer for detecting sulfa-5-methoxypyrimidine, wherein the sequence of the aptamer is SEQ No.1 recorded in a nucleotide sequence table: 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 the aptamer derivative obtained by modifying or reforming the aptamer.

Furthermore, the FAM fluorescent labeling aptamer sequence for detecting the sulfa-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 a FAM fluorophore.

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

1) Obtaining an aptamer of the sulfa-5-methoxypyrimidine through a fixed magnetic bead-SELEX screening method;

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

3) And (3) carrying out sequence truncation on the aptamer with the highest affinity through affinity measurement, so as to obtain a core recognition region of the aptamer, and obtaining the optimal aptamer.

The invention also providesFe-based detection of sulfa-5-methoxypyrimidine ₃ O ₄ /Au/g-C ₃ N ₄ The sensor composition comprising:

(1) FAM-SME4-1 described in the nucleotide sequence listing of FAM fluorescent markers;

(2)Fe ₃ O ₄ /Au/g-C ₃ N ₄ ；

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

The invention also provides a kit for detecting the sulfa-5-methoxypyrimidine, which comprises the Fe-based kit ₃ O ₄ /Au/g-C ₃ N ₄ Is provided.

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

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

(2)Fe ₃ O ₄ /Au/g-C ₃ N ₄ ；

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

(4) And (3) a magnet.

The application method of the kit comprises the following steps:

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

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

3) Adding Fe ₃ O ₄ /Au/g-C ₃ N ₄ Shaking is continued for 5 minutes;

4) After magnetic separation, the supernatant was aspirated and the fluorescence value was measured.

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

The invention provides a sulfonamide-5-methoxypyrimidine fluorescence detection kit, which comprises a fluorescent detection kit based onFe ₃ O ₄ /Au/g-C ₃ N ₄ Is a fluorescent aptamer sensor of (a); wherein the fluorescent aptamer sensor comprises Fe ₃ O ₄ /Au/g-C ₃ N ₄ And a FAM-labeled aptamer; the sequence of the FAM-marked aptamer is one of sulfa-5-methoxypyrimidine 4, sulfa-5-methoxypyrimidine 9, sulfa-5-methoxypyrimidine 15, sulfa-5-methoxypyrimidine 26, sulfa-5-methoxypyrimidine 50, sulfa-5-methoxypyrimidine 54 or sulfa-5-methoxypyrimidine 4-1 recorded in a nucleotide sequence table. The derivatives of the aptamer obtained after modification and transformation of the aptamer also belong to the protection scope of the invention.

The application of the sulfanilamide-5-methoxypyrimidine fluorescence detection kit provided by the invention comprises the following steps: the application of the sulfanilamide-5-methoxypyrimidine in quantitatively detecting 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 sulfa-5-methoxypyrimidine, and has the advantages of high sensitivity, simplicity in operation, strong selectivity, high speed and low cost. Has good application prospect in the detection of sulfanilamide-5-methoxypyrimidine.

2. According to the invention, the aptamer sequence obtained by magnetic bead-SELEX screening is analyzed and optimized to obtain the core recognition region of the aptamer, and the aptamer with high affinity and specificity to the sulfa-5-methoxypyrimidine is synthesized by in vitro cloning.

3. The invention relates to an aptamer based on sulfa-5-methoxypyrimidine and Fe ₃ O ₄ /Au/g-C ₃ N ₄ A fluorescent aptamer sensor was constructed. When the sulfa-5-methoxypyrimidine exists in the detection system, the aptamer specifically binds the sulfa-5-methoxypyrimidine and cannot be accumulated by Fe through pi-pi ₃ O ₄ /Au/g-C ₃ N ₄ And (5) adsorption. And the concentration of the sulfa-5-methoxypyrimidine is different, and the fluorescence value detected by the sulfa-5-methoxypyrimidine is also different. When the detection system does not contain sulfa-5-methoxypyrimidine, the fluorescent-labeled aptamer is accumulated by pi-pi to be coated with Fe ₃ O ₄ /Au/g-C ₃ N ₄ And (5) adsorption. Based on the principle, the quantitative detection of the sulfanilamide-5-methoxypyrimidine is realized.

4. Aiming at the problems that a traditional detection method cannot detect a large-scale sample, the operation is complicated, and long-time animal experiments are needed for preparing antibodies in an immunoassay method, the invention establishes a Fe-based method ₃ O ₄ /Au/g-C ₃ N ₄ The fluorescent sensor rapid detection method can be used for rapid and sensitive quantitative detection of sulfonamide-5-methoxypyrimidine residues in animal-derived foods, and overcomes the defects of the method. Provides a novel detection method for detecting antibiotics in animal-derived foods.

5. The invention is based on Fe ₃ O ₄ /Au/g-C ₃ N ₄ Screening and sequence optimizing the sulfanilamide-5-methoxypyrimidine aptamer, and constructing a Fe-based ₃ O ₄ /Au/g-C ₃ N ₄ The fluorescent aptamer sensor of the fluorescent aptamer sensor lays a foundation for the detection and development of products of sulfanilamide-5-methoxypyrimidine, and the fluorescent aptamer sensor successfully detects in actual sample milk and eggs and has good linear relation.

Drawings

Fig. 1: the invention is based on Fe ₃ O ₄ /Au/g-C ₃ N ₄ Schematic of fluorescent aptamer sensor principle;

fig. 2: determining aptamer affinity;

fig. 3: fluorescent aptamer sensor specificity analysis;

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

Detailed Description

EXAMPLE 1 establishment of a sulfanilamide-5-methoxypyrimidine aptamer detection System

1. Magnetic bead-SELEX process for screening sulfanilamide-5-methoxypyrimidine aptamer

1) Library and primer processing: 200. Mu.L of the 500nM aptamer library (5 '-FAM-CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTG C-3', N random sequence), 100. Mu.L of the 10. Mu.M forward primer (5'-CACCTAATACGACTCACTATAGCGGA-3') and 100. Mu.L of the 1. Mu.M reverse primer (5'-GCAAGCTTGTTCGAGCCAG-3') and 100. Mu.L of the 10. Mu.M Biotin-reverse primer (Biotin-5'-GCAAGCTTGTTCGAGCCAG-3') were placed in a 95℃metal bath for 10 minutes, followed by 30 minutes on ice, after which the library was taken out and stored at room temperature in the dark to allow a large amount of ssDNA to fold into a unique tertiary structure and the fluorescence value was determined.

2) Preparation of coupled magnetic beads and purified magnetic beads:

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

(2) 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 beads were stored in PBS (pH 7.4) at 4℃until use.

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

Collecting 800. Mu.L of the eluate and purifying the magnetThe beads were incubated at 25℃for 1 hour, after which time the beads were washed 2 times with PBS (pH 7.4) and the supernatant removed, then eluted with 50. Mu. L0.05M NaOH, and after 5 minutes incubation at 25℃the eluate (50. Mu.L) was collected by magnetic separation, and the pH was adjusted to 7.4 using 0.05M HCl. Then using 2 Xbinding buffer (175 mM NaCl, 4mM MgCl) ₂ 、40mM Tris-HCl、2mM CaCl ₂ 10mM KCl and 0.04%Tween 20,pH 7.6) equilibrates the buffer system in which the aptamer is located.

4) The ssDNA library obtained 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 2X taq PCR MasterMix II and 36. Mu.L of ddH were mixed ₂ O-mix, denaturation at 95℃for 30s, renaturation at 58℃for 30 seconds, and extension at 72℃for 6 seconds. The amplification process was repeated 20 rounds. The amplified product was purified using the same procedure as in step 3) above and used as an aptamer library for the next round.

5) The seventh round uses sulfadimidine, sulfaquinoxaline and sulfadiazine-magnetic beads for reverse screening, and then uses sulfa-5-methoxypyrimidine-magnetic beads for continuous forward screening. Ultraviolet-visible spectrum UV-VIS spectrum was used to determine the concentration of aptamer at the end of each round of screening.

6) Determination of recovery: (concentration of ssDNA recovered at the end of each round of purification/concentration of ssDNA dosed at the beginning of each round) ×100%.

2. Cloning and sequencing

The product obtained by ten rounds of screening is amplified by PCR, and the cloned product is selected for sequencing.

3. Aptamer sequence analysis and optimization

Predicting the secondary structure of the resulting aptamer by DANMAN, RNAstructure, viennaRNA Web Services and UNAFold Web Server; the sequences meeting the requirements are selected and shown in Table 1. The affinity of the candidate aptamer is then determined by an affinity assay. Sequence cutting is carried out on the aptamer with highest affinity, so that a new aptamer is obtained: SME4-1, sequences are shown in Table 2.

TABLE 1

TABLE 2

4. Affinity assay

To assess the affinity of candidate aptamers, 100 μl of SME-carboxyl functionalized magnetic beads (10 mg/mL) were mixed with 200 μl of FAM-labeled aptamers at different concentrations (i.e. 0, 25, 50, 100, 200 and 400 nM), the mixture was reacted in the dark at 25 ℃ for 1 hour, and then the reaction mixture was washed twice with binding buffer. Subsequently, 100. Mu.L of 50mM NaOH was added to the resulting beads, and incubated for 5 minutes with gentle stirring. Finally, the supernatant from the reaction mixture was collected and applied to a fluorometer at λ _ex =492 nm and λ _em Its fluorescence intensity was measured at=518 nm. K representing binding affinity _d Values were analyzed using Origin 2019 software and according to the nonlinear regression equation y=b _max ×X÷(K _d +X) calculation, wherein X represents the aptamer concentration, Y represents the relative fluorescence intensity, B _max Representing 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 Table 3

The aptamer derivative obtained by modifying and transforming the aptamer also belongs to the protection scope of the invention.

5.Fe ₃ O ₄ /Au/g-C ₃ N ₄ Is prepared from

15 g of urea are heated at 550℃for 4 hours, at the end of which a yellow g-C is obtained ₃ N ₄ . Will be 0.5g g-C ₃ N ₄ Added to 25mL of trisodium citrate solution (1.5 mM) and sonicated for 20 minutes. To the reaction mixture was added 25mL of chloroauric acidSolution (1 mM) 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 and absolute ethanol, respectively, and then dried at 60℃to obtain Au/g-C ₃ N ₄ . 0.15 g Au/g-C ₃ N ₄ 、0.135g FeCl ₃ ·6H ₂ O and 0.2025g FeSO ₄ ·7H ₂ O was dissolved in 50mL DI H ₂ In O, sonicate for 1 hour. Subsequently, 0.12g of NaOH was dissolved in the solution, 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 for 4 times to obtain brown-black magnetic material Fe ₃ O ₄ /Au/g-C ₃ N ₄ 。

6. Specific analysis of aptamer and sulfa-5-methoxypyrimidine

To evaluate the aptamer specificity for sulfa-5-methoxypyrimidine, 200 μl of a reaction mixture was prepared, wherein the concentration of the fluorescently labeled aptamer was 100nM, and the final concentration of sulfa-5-methoxypyrimidine or its structural analog (sulfaquinoxaline, sulfadiazine, sulfadimidine, sulfamethoxazole, sulfapyridine, nitrofurantoin, ethanamine, and kanamycin) was 1 μg/μl. 30. Mu.L of 2mg/mL Fe ₃ O ₄ /Au/g-C ₃ N ₄ Adding into the mixture, mixing, and adsorbing Fe with magnet ₃ O ₄ /Au/g-C ₃ N ₄ I.e., after magnetic separation, the supernatant was aspirated and the fluorescence value was measured (as shown in FIG. 1). The specificity of the aptamer was evaluated using a fluorescence intensity comparison, the formula being fluorescence intensity comparison = (measured fluorescence intensity of other antibiotics/measured fluorescence intensity of sulfa-5-methoxypyrimidine) ×100%. As shown in FIG. 3, the relative fluorescence intensity of other antibiotics and sulfonamide-5-methoxypyrimidine structural analogues was less than 25%. These results indicate that SME4-1 has higher specificity for sulfa-5-methoxypyrimidine.

7. Establishment of a Standard Curve

The standard curve was constructed using a sulfonamide-5-methoxypyrimidine standard, and FAM fluorescent-labeled aptamer (100 nM) was conjugated to a series of sulfonamide-5-methoxypyrimidine concentrations of 2-250ng/mL at 200. Mu.LIncubating in buffer solution at 25deg.C in dark for 1h, fe ₃ O ₄ /Au/g-C ₃ N ₄ To the mixture, incubated for 5 minutes in the dark at room temperature, the fluorescence intensity was measured by a microplate reader at an emission wavelength of 520nm, and a standard curve was drawn (shown in FIG. 4).

EXAMPLE 2 determination of actual sample sulfa-5-methoxypyrimidine

The aptamer sensor was validated using 10 different samples purchased from the local market, after which the samples were confirmed to be free of sulfa-5-methoxypyrimidine using HPLC. 10mL of skim milk was centrifuged at 14000rpm for 20 minutes at 4℃and the supernatant was collected and diluted to 100mL. Finally, the mixture was filtered through a 0.22 μm filter. 2g of eggs and 4ml of ethyl acetate were shaken for 10 minutes, centrifuged at 5000 rpm for 5 minutes and finally the ethyl acetate was removed with a metal bath nitrogen blower at 40 ℃. The honey was diluted 10-fold with PBS to eliminate matrix interference. Mashing pig, pig liver, chicken liver, cattle, carassius auratus and shrimp with a meat grinder, and homogenizing with a homogenizer to obtain homogenized emulsifying agent (for Carassius auratus, peeling, and for shrimp, removing shell, and digestive tract of shrimp, only shrimp meat is taken). 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 fat. The liquid of 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 sulfa-5-methoxypyrimidine (50, 100 and 150 μg/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/standard concentration). Times.100%. The coefficient of variation was determined by analyzing the above-described samples to which three different levels of sulfa-5-methoxypyrimidine were added. The calculation formula of the variation coefficient is as follows: coefficient of variation cv= (standard deviation/average value) ×100%. Each concentration level was tested five times and the correlation between the aptamer sensor and the HPLC analysis in terms of detection of sulfa-5-methoxypyrimidine in the labeled sample was calculated.

As shown in Table 4, the recovery of the samples was between 92.42% and 107.22%, and the coefficient of variation was between 1.43% and 14.46%. A positive correlation was also observed between the aptamer sensor and the results of HPLC (R ² >0.9304). The results indicate that the proposed aptamer sensor is used for detecting the reliability of sulfa-5-methoxypyrimidine.

Table 4: average recovery and coefficient of variation of the labeled samples, correlation between aptamer sensor and HPLC results (n=5).

Example 3 the invention provides a kit for detecting sulfa-5-methoxypyrimidine:

the kit comprises the following components:

(1) FAM-SME4-1 described in the 100nM FAM fluorescence-labeled nucleotide sequence listing;

(2)2mg/mLFe ₃ O ₄ /Au/g-C ₃ N ₄ ；

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

(4) And (3) a magnet.

The detection is carried out according to the following steps:

1) Treating each animal-derived food sample according to the requirements;

2) The treated sample was dissolved in the binding buffer and diluted 10-fold. 100nM FAM-SME4-1 was added and mixed and shaken for 30 min;

3) 30 mu L of 2mg/mL Fe was added ₃ O ₄ /Au/g-C ₃ N ₄ Shaking is continued for 5 minutes;

4) The magnet adsorbs Fe ₃ O ₄ /Au/g-C ₃ N ₄ I.e. after magnetic separation, the supernatant is aspirated and the fluorescence value is measured.

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

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

SEQUENCE LISTING

<110> Chongqing university

Shenzhen Institute of Information Technology

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

<130> CN 107119054 A

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 90

<212> DNA

<213> aptamer

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cacctaatac gactcactat agcggatccg actggctcga acaccgttac cccttagtcc 60

tgcgtctgtt cctggctcga acaagcttgc 90

<210> 2

<211> 90

<212> DNA

<213> aptamer

<400> 2

cacctaatac gactcactat agcggatccg atctggctcg aacgcaatgc acgaaaatta 60

ggatgtctgg cctggctcga acaagcttgc 90

<210> 3

<211> 90

<212> DNA

<213> aptamer

<400> 3

cacctaatac gactcactat agcggatccg atacgtctcg taagggtcaa gtatggcata 60

gaccgacttc cctggctcga acaagcttgc 90

<210> 4

<211> 90

<212> DNA

<213> aptamer

<400> 4

cacctaatac gactcactat agcggatccg aactctacct agtacctggc tcgaccctgg 60

ctcgaacact cgctggctcg aacaagctgc 90

<210> 5

<211> 90

<212> DNA

<213> aptamer

<400> 5

cacctaatac gactcactat agcggatccg aacattgtag tggctcgaac aacgttactg 60

gccgtgaact cctggctcga acaagcttgc 90

<210> 6

<211> 90

<212> DNA

<213> aptamer

<400> 6

cacctaatac gactcactat agcggatccg atacgaactg ggttgaacac cctactaaac 60

tggctcatat gctggctcga acaagcttgc 90

<210> 7

<211> 13

<212> DNA

<213> aptamer

<400> 7

ccgactggct cgg 13

Claims

1. A FAM fluorescent-labeled aptamer for detecting sulfa-5-methoxypyrimidine, characterized in that: the nucleotide sequence of the aptamer is shown as SEQ No.1, SEQ No.2, SEQ No.3, SEQ No.4, SEQ No.5, SEQ No.6 or SEQ No. 7.

2. The FAM fluorescent-labeled aptamer for detecting sulfa-5-methoxypyrimidine according to claim 1, wherein: the aptamer consists of single-stranded DNA, and the 5' end is labeled with a FAM fluorophore.

3. Based on Fe ₃ O ₄ /Au/g-C ₃ N ₄ Is characterized in that the sensor composition comprises:

(1) Nucleotide with FAM fluorescent label sequence shown as SEQ No.7, (2) Fe ₃ O ₄ /Au/g-C ₃ N ₄ ；

(3) Binding buffer solution.

4. A kit for detecting sulfa-5-methoxypyrimidine, characterized in that: comprising the Fe-based alloy according to claim 3 ₃ O ₄ /Au/g-C ₃ N ₄ Is provided.

5. The kit for detecting a sulfa-5-methoxypyrimidine of claim 4, wherein: the kit comprises the following components:

(1) FAM fluorescent labeling sequence is shown as the nucleotide shown in SEQ No.7,

(2)Fe ₃ O ₄ /Au/g-C ₃ N ₄ ；

(3) Binding buffer solution;

(4) And (3) a magnet.

6. The method of using the kit of claim 5, wherein: the method comprises the following steps:

1) Treating each animal-derived food sample according to the requirements;

2) Mixing the treated sample with FAM fluorescent labeled nucleotide with the sequence shown in SEQ No.7, and shaking for 30 minutes;

3) Adding Fe ₃ O ₄ /Au/g-C ₃ N ₄ Shaking is continued for 5 minutes;

7. The use of the kit for detecting sulfa-5-methoxypyrimidine of claim 5 for quantitatively detecting sulfa-5-methoxypyrimidine in animal-derived tissue test samples.

8. The use according to claim 7, wherein the animal derived tissue test sample is milk, honey, egg, pork, beef, chicken, pork liver, chicken liver, crucian or shrimp.